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Molluscan 
Research 


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The Malacological Society of Australasia 


Molluscan Research 


Molluscan Research is a publication for authoritative scientific papers dealing with the 
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Molluscan Research 


Contents Volume 23 Number 2 2003 


Embryonic and larval development of Pinctada margaritifera (Linnaeus, 1758) 
M. S. Doroudi and P. C. Southgate 101 


Ultrastructure of male germ cells in the testes of abalone, Haliotis ovina Gmelin 
S. Singhakaew, V. Seehabutr, M. Kruatrachue, P. Sretarugsa and S. Romratanapun “109 


Gastropod phylogeny based on six segments from four genes representing coding or non- 
coding and mitochondrial or nuclear DNA 
D. J. Colgan, W. F. Ponder, E. Beacham and J. M. Macaranas 123 


Reassessment of Australia's oldest freshwater snail, Viviparus (?) albascopularis 
Etheridge, 1902 (Mollusca : Gastropoda : Viviparidae), from the Lower Cretaceous 
(Aptian, Wallumbilla Formation) of White Cliffs, New South Wales 

B. P. Kear, R. J. Hamilton-Bruce, B. J. Smith and K. L. Gowlett-Holmes 149 


Relationships of Placostylus from Lord Howe Island: an investigation using the 
mitochondrial cytochrome c oxidase 1 gene 
W. F. Ponder, D. J. Colgan, D. M. Gleeson and G. H. Sherley 159 


Short contribution 


Changes in tissue composition during larval development of the blacklip pearl oyster, 
Pinctada margaritifera (L.) 
J. M. Strugnell and P. C. Southgate 179 


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CSIRO PUBLISHING 
www.publish.csiro.au/journals/mr Molluscan Research, 2003, 23, 101—107 


Embryonic and larval development of Pinctada margaritifera 
(Linnaeus, 1758) 


Mehdi S. Doroudi^P€ and Paul C. Southgate^ 


^School of Marine Biology and Aquaculture, James Cook University, Townsville, Qld 4811, Australia. 
BNSW Fisheries, c/o Murray Irrigation, Wakool, NSW 2710, Australia. 
CAuthor to whom all correspondence should be addressed. Email: mehdi.doroudi@fisheries.nsw.gov.au 


Abstract 


We describe the developmental stages of the black-lip pearl oyster, Pinctada margaritifera (Linnaeus, 
1758), larvae from fertilisation through embryonic development and larval growth in the laboratory at 
28 + 1°C. Larvae were anesthetised, fixed, critical-point dried and examined using a scanning electron 
microscope. We examined embryonic development (fertilisation, polar body, blastomeres, gastrula) and 
attributes of the larval shell (size, prodissoconch I/II, growth lines, provinculum, shell fracture) and larval 
velum. The first polar body formed 24 min after fertilisation and fertilised eggs had a mean diameter of 
59.9 + 1.4 um. The earliest actively swimming trochophore appeared 8-12 h after fertilisation. The D stage 
was reached approximately 24 h after fertilisation and measured 79.7 + 2.3 um in shell length. Ten-day-old 
larvae had umbones that arose opposite each other above the hinge axis and 22-day-old larvae, with a mean 
shell length of 230.8 + 4.9 um, developed a pigment spot just before entering the pediveliger stage. 


Additional keywords: hinge, morphology, pearl oyster, scanning electron microscope. 


Introduction 


Herdman (1903) studied the early life stages of Pinctada vulgaris (= P. fucata Gould, 1850) 
larvae up to 3 days after fertilisation. Other studies have investigated larval development 
and growth of P. fucata, P. martensi Dunker, 1850 and P maxima Jameson, 1901 (Ota 1957; 
Minaur 1969; Tanaka and Kumeta 1981; Alagarswami et al. 1983). Rose and Baker (1994) 
described larval development of P. maxima in detail and compared their findings with those 
reported for other pearl oyster species. Although P margaritifera larvae have been cultured 
since 1970 (Tanaka et al. 1970; Alagarswami et al. 1989; Southgate and Beer 1997), 
embryonic development and the morphology of different larval stages have not been 
described in detail. 

The present study notes the characteristics of P. margaritifera larvae that have not been 
reported on previously using scanning electron microscopy (SEM) and provides a basic 
understanding of larval development during hatchery culture. 


Materials and methods 
Larval rearing 


Pinctada margaritifera broodstock were induced to spawn by thermal stimulation and the addition of sperm 
in a seawater suspension. Fertilised eggs were stocked at a density of 30 mL”! in aerated fibreglass tanks 
(500 L) filled with 1 um filtered seawater at 28°C. The salinity of seawater was 33, which was measured 
using the practical salinity scale. After 24 h, when D-stage larvae (shell becomes D-shaped) had a mean 
shell length of 79.7 + 2.3 um, they were collected on a 25-um mesh sieve, counted and placed at a density 
of 2 mL! in 500-L aerated fibreglass tanks containing filtered seawater at the same temperature and 
salinity. 

We cultured Tahitian /sochrysis aff. galbana Green (T-ISO) and Pavlova salina Green in 3-L glass flasks 
and 20-L carboys in autoclaved 0.45-m-filtered and ultraviolet (UV)-treated seawater using f/2 nutrient 


© Malacological Society of Australasia 2003 10.107 I/MR02015 1323-5818/03/020101 


102 Molluscan Research M. S. Doroudi and P. C. Southgate 


medium (Guillard 1983). Microalgae cultures were provided with illumination from cool white fluorescent 
lights with a 12-h light: 12-h dark photoperiod. Larvae were fed daily a 1:1 mixture of T-ISO and Pavlova 
salina at a ration of 1-18 x 10? cells mL”1 (Southgate and Beer 1997, Doroudi er al. 1999a). We conducted 
three separate spawnings to collect embryonic and larval samples. 


Sample preparation 


We observed embryonic development every 15 min during the first 3 h, then once an hour until the 
trochophore stage (8-24 h) and D-stage (24 h) using a compound microscope. Larvae were narcotised in a 
15% (w/v) solution of MgCl, (Bellolio et al. 1993) and seawater (1:1) at 28°C for 5-10 min. We collected 
samples from three tanks at 2-day intervals after the D stage (24 h) and fixed them in 2.5% glutaraldehyde 
in 0.1 M piperazine at pH 7.6. This sampling continued until larvae had developed to the “eyed” stage (i.e. 
when larvae develop a pigment spot). Subsamples of larvae were post-fixed with osmium tetroxide (OsO,), 
dehydrated in a graded series of ethanol, critical-point dried in liquid carbon dioxide (CO,), mounted on 
aluminium stubs with double-sided tape and coated with gold before being examined using an SEM. 

We collected larvae on a mesh sieve, washed them into a graduated cylinder and removed a subsample, 
from which the shell length of 40 larvae was measured using a compound microscope. Morphological 
terminology follows that commonly used for bivalve larvae in similar studies (Waller 1981; Belollio et al. 
1993). 


Results 


The time series of developmental stages of P margaritifera embryos and larvae is shown in 
Table 1. 


Embryo to trochophore 


Unfertilised P. margaritifera eggs had a mean diameter of 39.7 + 1.3 um (n = 40; Fig. 1). 
The first polar body formed 24 min after fertilisation and fertilised eggs had a mean 
diameter of 59.9 + 1.4 um (n = 40). The four blastomeres resulting from the second cleavage 
formed 2 h after fertilisation. Cell division followed the usual bivalve pattern for bivalves 
and resulted in the gastrula, 5 h after fertilisation. The change from a ciliated gastrula to the 
trochophore stage was gradual and the earliest actively swimming trochophore appeared 
8—12 h after fertilisation. Morphological changes from trochophore to the D stage included 
extension along the longitudinal axis and the apical region becoming broader than the 
posterior region. At this time, cilia on the apical region became longer. With the 
development of long cilia, larvae began to secrete shell and the resulting larvae swam 
actively using the velum. 


Larvae 


The D stage was reached approximately 24 h after fertilisation and larvae measured 79.7 
+ 2.3 um (n = 40) in shell length. The D-stage larvae showed preliminary growth rings after 
2 days (Fig. 2). The shell showed slight umbonal growth after 6 days development and 
prodissoconch I and II could be clearly identified (Fig. 3). At a shell length of 
approximately 100 um, the hinge developed denticulation on either side of a central region. 
As the larva grew, the hinge developed and formed a series of teeth and sockets on each 
valve (Fig. 4). Each valve had teeth on either side of a central area (Fig. 5). The central area 
included a series of tiny teeth and sockets (Fig. 6). Ten-day-old larvae had umbones that 
arose opposite each other above the hinge axis (Fig. 7) and 22-day-old larvae, with a mean 
shell length of 230.8 + 4.9 um (n = 40), developed a pigment spot and entered the 
pediveliger stage shortly after (Fig. 8). Sections of broken shell edges at this stage suggest 
that calcification of the shell by the mantle had occurred in prodissoconch II (Fig. 9). 
Fractures through prodissoconch II showed layering in the shell structure and indicated that 
the shell is thicker in the area of growth lines (Fig. 10). The oval velum was located at the 


103 


Molluscan Research 


Development of Pinctada margaritifera 


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104 Molluscan Research M. S. Doroudi and P. C. Southgate 


Figs 1-6. 7, Unfertilised egg of Pinctada margaritifera with sperm (s) on the surface. 2, External view 
of the right valve of Pinctada margaritifera D-stage larvae; I, prodissoconch I; II, prodissoconch II. 3, 
Prodissoconch I (I) and II (II) in the early umbo stage of Pinctada margaritifera larvae. 4, Development 
of the provinculum of Pinctada margaritifera larvae (umbo stage); t, tooth; c, central area. 5, Main teeth 
on either side ofa central area of Pinctada margaritifera larvae (umbo stage); t, tooth; s, socket. 6, Central 
area of the Pinctada margaritifera larval hinge (umbo stage) showing the series of teeth (t) and sockets (s). 


Development of Pinctada margaritifera i Molluscan Research 105 


Fig. 7-12. 7, Hinge length (h) and umbones (u) arise over the hinge axis of Pinctada margaritifera 
larvae (umbo stage). 8, An eyed larva of Pinctada margaritifera showing details of umbonal features (u); 
r, right valve. 9, The mantle (m) viewed from a cross-section of the prodissoconch II (IT) of Pinctada 
margaritifera larvae (umbo stage). 10, Fracture of the prodissoconch in Pinctada margaritifera umbo 
larva; p, outer prismatic layer; gh, granular homogeneous layer. /1, Lateral view of the entire Pinctada 
margaritifera D-stage larva with velum (v) extended at the anterior dorsal side of the shell; r, right valve. 
12, Enlargement of cilia (c) on the velum of Pinctada margaritifera D-stage larvae. 


posterior dorsal side of the larva (Fig. 11) and was well developed with a peripheral ring of 
cilia (Fig. 12). 


Discussion 


Pinctada margaritifera larvae need a period of 8 days to reach the early umbo stage (shell 
length 110 um) and exhibit an average daily growth rate of 3.7 um. Elsewhere, a daily 


106 Molluscan Research M. S. Doroudi and P. C. Southgate 


growth rate of 5 um has been reported for P. margaritifera during the first 7 days of the 
larval rearing period (Tanaka ez al. 1970). Growth rates of bivalve larvae are likely to be 
influenced by genetic factors, as well as endogenous and exdogenous nutrition and culture 
conditions. Because P. margaritifera larvae have exponential growth (Doroudi et al. 
19992), the mean daily growth rate increased up to 7.2 um over the period of 22 days 
required for larvae to reach the eye spot stage (230 um). Eye spots in P. margaritifera 
generally occur in larvae that are 230 um or greater in shell length. In a previous study, 
P. margaritifera larvae developed a pigment spot at 210 um shell length (Alagarswami et al. 
1989). In P. fucata (Alagarswami et al. 1983) and P. maxima (Rose and Baker 1994), eye 
spots form in individuals that are approximately 210 and 230 um in shell length, 
respectively. Despite variations in rearing conditions (e.g. environmental factors, type of 
food and genetic differences), P. margaritifera, P. fucata and P. maxima settle at 
approximately the same size (230-266 um) and age (20—23 days) from fertilisation (Rose 
and Baker 1994). The overall development of P. margaritifera larvae described in the 
present study is similar to the more general descriptions of pearl oyster larvae reported in 
previous studies (Table 1). 

The larvae of bivalves are similar in exterior appearance and are difficult to differentiate 
without detailed anatomical study. The present SEM study of P. margaritifera larvae 
revealed some anatomical features of the larval shell that have not been observed previously 
using other techniques. For instance, the punctate region on the exterior surface of 
prodissoconch I of P. margaritifera is also observed in Crassostrea virginica Gmelin, 1791 
(Carriker and Palmer 1979) and Ostrea edulis Linnaeus, 1750 (Waller 1981). Hinge 
structure can be a primary character in identification of bivalve larvae (Le Pennec 1980). 
Lutz et al. (1982) reported that hinge structure differed among 12 genera of bivalves, 
whereas the basic hinge morphology of P. margaritifera seems to be similar to that of other 
pearl oysters; that is, a tooth and socket at each end with a thin central area. The present 
study has shown that 8-day-old larvae of P. margaritifera, with a shell length of 110 um, 
have five teeth in each valve, with three at the anterior end of the hinge line and two at the 
posterior. This is the same as reported for P. maxima larvae with a shell length of 90 um 
(Rose and Baker 1994). Our observations in the present study on the larval rearing of 
P. margaritifera provide a basic understanding of larval development during hatchery 
culture of this species. 


Acknowledgments 


This study was partially funded by the Australian Centre for International Agricultural 
Research (ACIAR) as part of Project No. FIS/97/31 *Pearl Oyster Resource Development 
in the Pacific Islands'. 


References 


Alagarswami, K., Dharmaraj, S., Velayudhan, T. S., Chellam, A., Victor, A. C. C., and Gandhi, A. D. 
(1983). Larval rearing and production of spat of pearl oyster Pinctada fucata (Gould). Aquaculture 34, 
287-301. 

Alagarswami, K., Dharmaraj, S., Chellam, A., and Velayudhan, T. S. (1989). Larval and juvenile rearing of 
black-lip pearl oyster Pinctada margaritifera (Linnaeus). Aquaculture 76, 43—56. 

Bellolio, G., Lohrmann, K., and Dupre, E. (1993). Larval morphology of the scallop Argopecten purpuratus 
as revealed by scanning electron microscopy. The Veliger 36, 332-342. 

Carriker, M. R., and Palmer, R. E. (1979). Ultrastructural morphogenesis of prodissoconch and early 
dissoconch valves of the oyster Crassostrea virginica. Proceedings of the National Shellfisheries 
Association 69, 103—128. 


Development of Pinctada margaritifera Molluscan Research 107 


Doroudi, M. S., Southgate, P. C., and Mayer, R. (1999a). The combined effects of temperature and salinity 
on the embryo and larvae of black-lip pearl oyster, Pinctada margaritifera (L). Aquaculture Research 
30, 271—277. 

Doroudi, M. S., Southgate, P. C., and Mayer, R. (19992). Growth and survival of the black-lip pearl oyster 
(Pinctada margaritifera, L.) larvae fed at different algal density. Aquaculture International 7, 179—187. 

Guillard, R. L. (1983). Culture of phytoplankton for feeding marine invertebrates. In *Culture of Marine 
Invertebrates’. (Ed. C. L. Berg.) pp. 108-132. (Hutchinson Ross: Stroudberg.) 

Herdman, W. A. (1903). Observations and experiments on the life-history and habits of the pearl oyster. In 
*Report Pearl Oyster Fisheries, Gulf of Mannar'. (Ed. W. A. Herdman.) pp. 125-146. (Royal Society: 
London.) 

Le Pennec, M. (1980). The larval and post-larval hinge of some families of bivalve mollusks. Journal of 
Marine Biology Association UK 60, 601—617. 

Lutz, R., Goodsell, M., Castagna, M., Chapman, S., Newell, C., Hidu, H., Mann, R., Jablonski, D., Kennedy, 
V, Siddall, S. et al. (1982). Preliminary observations on the usefulness of hinge structures for 
identification of bivalve larvae. Journal of Shellfish Research 2, 65—70. 

Minaur, J. (1969). Experiments on the artificial rearing of the larvae of Pinctada maxima (Jameson) 
(Lamellibranchia). Australian Journal of Marine and Freshwater Research 20, 175—187. 

Ota, S. (1957). Notes on the identification of free swimming larvae of pearl oyster (Pinctada martensii). 
Bulletin of the National Pearl Research Laboratory, Japan 2, 128-132. 

Rose, R. A., and Baker, S. B. (1994). Larval and spat culture of the Western Australian silver or gold lip 
pearl oyster, Pinctada maxima (Jameson) (Mollusca: Pteriidae). Aquaculture 126, 35-50. 

Southgate, P. C., and Beer, A. C. (1997). Hatchery and early nursery culture of the blacklip pearl oyster 
(Pinctada margaritifera, L.). Journal of Shellfish Research 16, 561—568. 

Tanaka, Y., and Kumeta, M. (1981). Successful artificial breeding of the silver-lip pearl oyster, Pinctada 
maxima (Jameson). Bulletin of the National Research Institute of Aquaculture, Japan 2, 21-28. 

Tanaka, Y., Inoha, S., and Kakazu, K. (1970). Studies on seed production of black-lip pearl oyster Pinctada 
margaritifera, in Okinawa. V. Rearing of the larvae. Bulletin of Tokai Region Fisheries Research 
Laboratory, Japan 63, 97—106. 

Waller, T. R. (1981). Functional morphology and development of veliger larvae of the European oyster, 
Ostrea edulis Linne. Smithsonian Contributions to Zoology 328, 1—70. 


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CSIRO PUBLISHING 
www.publish.csiro.au/journals/mr Molluscan Research, 2003, 23, 109—121 


Ultrastructure of male germ cells in the testes of abalone, 
Haliotis ovina Gmelin 


Sombat Singhakaew^, Viyada Seehabutr®, Maleeya Kruatrachue^, Prapee Sretarugsa© 
and Suppaluk Romratanapun® 


‘Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. 
Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10400, Thailand. 
“Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. 
To whom correspondence should be addressed. Email: scmkt@mahindol.ac.th 


Abstract 


An ultrastructural study of male germ cells in the testes of Haliotis ovina revealed that spermatogenesis 
could be classified into 13 stages, based on the pattern of chromatin condensation and distribution of 
organelles, as follows: the spermatogonium; five stages of the primary spermatocyte; the secondary 
spermatocyte; five stages of the spermatid; and the spermatozoa. Each spermatogonium was round or oval, 
with a euchromatic nucleus and prominent nucleolus. The primary spermatocytes were divided into five 
stages: leptotene (LSc); zygotene (ZSc); pachytene (PSc); diplotene (DSc); and metaphase (MSc). The 
nucleus of the LSc contained scattered small heterochromatin blocks that were increasingly thickened in 
the ZSc. The PSc was characterised by a bouquet pattern of heterochromatin fibres. The DSc decreased in 
size, resulting in close clumping of chromatin blocks, whereas in the MSc, long and large blocks of 
chromosomes were formed and then moved to be aligned along the equatorial region. Secondary 
spermatocyte showed thick chromatin blocks that appeared reticulate. The spermatid could be divided into 
five stages (St,_;). The St, was a large round cell and its nucleus contained homogeneous chromatin 
granules. In St), the nuclear chromatin started to condense into patches. The St; was smaller with a round 
nucleus containing dark blocks of heterochromatin. The St, became smaller still, with a round opaque 
nucleus. The St; was the smallest round cell, with almost completely condensed chromatin. The 
spermatozoon had a round to barrel-shaped head that contained completely condensed chromatin covered 
by a conical acrosome. The posterior border of the nucleus was flanked by five large spherical mitochondria 
and the tail consisted of axonemal microtubules surrounded by the plasma membrane. 


Introduction 


There are almost 100 species of abalone all belonging to the genus Haliotis (Fallu, 1991). 
These snails are widely distributed in tropical and temperate seas and inhabit submerged 
rock (Crofts 1929). Since ancient times, the abalone has been an economically important 
snail because of its decorative shell and food value. Abalone are commercially important 
molluscs in many countries, such as Japan, America, Mexico and Australia, where the 
culture of commercial abalone is well established. In Thailand, there are three species of 
abalone, namely Haliotis ovina Gmelin, 1791, H. asinina Linnaeus, 1758 and H. varia 
Linnaeus, 1929 (Nateewathana and Hylleberge 1986; Tookvinart ef al. 1986; Bussarawit 
et al. 1990). Of these the three species, H. ovina is the one with economic potential 
(Nateewathana and Hylleberge 1986; Bussarawit et al. 1990). 

Species of Haliotis are dioecious, with clear sexual dimorphism. The single testis 
envelops the digestive gland and, together, these structures form a large cone-shaped 
appendage called the conical organ, which wraps around the right and posterior margins of 
the shell muscle (Crofts 1929). The colour of the gonad indicates the sex of the abalone. 
The testis is cream to ivory, whereas the ovary is green in colour (Singhagraiwan 1989). 


© Malacological Society of Australasia 2003 10.1071/MR02016 1323-5818/03/020109 


110 Molluscan Research S. Singhakaew et al. 


There is no genital duct in Haliotis (Crofts 1929). The sperm from the testis are expelled 
into the cavity of the right renal organ, which is seen on the dorsal surface of the body and 
is overlapped by the digestive gland, and finally pass through shell perforations, which are 
located on the left side (Fallu 1991), into the water. 

There have been extensive ultrastructural studies of male germ cells, especially the 
spermatozoa, in several haliotid species, such as Haliotis rufescens Swainson, 1822 (Lewis 
et al. 1980), H. aquatilis Reeve, 1846 (Shiroya and Sakai 1992), H. diversicolor supertexta 
Lischke, 1870 (Gwo et al. 1997), H. discus Reeve, 1846 (Sakai et al. 1982; Usui 1987), 
H. midae Linnaeus, 1758 (Hodgson and Foster 1992), H. laevigata Donovan, 1808 (Healy 
et al. 1998), and H. asinina (Apisawetakan et al. 2000; Sobhon et al. 2001). In general, 
spermatogenesis in these haliotid species can be classified into the following stages: 
spermatogonia; primary spermatocytes; secondary spermatocytes; spermatids; and 
spermatozoa. Sobhon et al. (2001) differentiated male germ cells in H. asinina into 14 
stages based on the ultrastructure and pattern of chromatin condensation. These stages were 
the spermatogonium, six stages of the primary spermatocyte, the secondary spermatocyte, 
four stages of the spermatid and the spermatozoa (immature and mature). The general 
ultrastructure of haliotid spermatozoa is typical of the primitive type described for molluscs 
that reproduce by external fertilisation. The spermatozoa have a very simplified midpiece 
that is composed of a cluster of spherical mitochondria surrounding a pair of orthogonally 
arranged centrioles (Lewis et al. 1980; Sakai er al. 1982; Hodgson and Bernard 1986; 
Hodgson and Foster 1992; Shiroya and Sakai 1992; Gwo et al. 1997; Healy et al. 1998; 
Apisawetakan et al. 2000). 

In the present study, we examined the ultrastructure of male germ cells in H. ovina, an 
abalone of potential economic importance, which is common along the coast of Thailand. 
The results are compared with those of H. asinina and other abalone species. 


Materials and methods 


Adult H. ovina (approximately 7 cm shell length, 170 g weight) were collected during June and July 1999 
from Samed Island, Rayong Province, Thailand. A total of 20 male abalone was used in the present study. 
For the ultrastructural study, very small pieces of testes were fixed in 306 glutaraldehyde in 0.1 M sodium 
cacodylate buffer (pH 7.8) at 4°C overnight, washed in 0.1 M sodium cacodylate buffer and post-fixed in 
1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at 4°C. The pieces were then dehydrated in 
a graded series of ethanol, cleared using propylene oxide and infiltrated and embedded in Araldite 520 
epoxy resin. Sections were cut with glass knives on a Sorvall MT-2 ultramicrotome. Semithin sections were 
stained with toluidine blue and observed with a light microscope. Ultrathin sections were stained with 
uranyl acetate and lead citrate and were viewed with a Hitachi H300 transmission electron microscope 
(TEM) operated at 75 kV. 


Results 


The male germ cells of H. ovina can be classified into 13 stages based on cell shape and 
size, nuclear shape and size, and pattern of chromatin condensation (Fig. 1). These stages 
comprise the spermatogonium, five stages of the spermatocyte, the secondary 
spermatocyte, five stages of the spermatid and the spermatozoa. 


Spermatogonium 


These cells lie on the outer edges of the lobe of the testis. The spermatogonium is spherical 
or oval, with a diameter of approximately 8-10 um (Fig. 14,3). The nucleus is round or 
slightly oval and contains mostly euchromatin, with only a thin rim of heterochromatin 
blocks attached to the inner surface of the nuclear envelope (Fig. 24). The nucleolus is 


Male germ cells of abalone Molluscan Research 111 


Fig. 1. Photomicrograph (A) and electron micrographs (B-D) showing various stages of the male germ 
cells of Haliotis ovina. Sg, Spermatogonia; LSc, leptotene primary spermatocyte; ZSc, zygotene primary 
spermatocyte; PSc, pachytene primary spermatocyte; DSc, diplotene primary spermatocyte; SSc, 
secondary spermatocyte; St, spermatid; Sz, spermatozoa. 


prominent against the background of a rather transparent nucleoplasm. The cytoplasm 
contains free ribosomes and numerous mitochondria, which appear at the outer surface of 
the nuclear envelope (Fig. 24). 


Spermatocytes 


The primary spermatocytes (PrSc) consist of five stages: leptotene (LSc), zygotene (ZSc), 
pachytene (PSc), diplotene (DSc) and metaphase (MSc) spermatocytes. The early cells 


112 Molluscan Research S. Singhakaew et al. 


Fig.2. A, High magnification of a spermatogonium (Sg) showing the round nucleus (Nu) with distinct 
nucleolus (No) and heterochromatin (hc) blocks. Mitochondria (Mi) are numerous on the outer surface 
of the nuclear envelope (NE). B, A leptotene primary spermatocyte (LSc) contains an oval nucleus with 
nucleolus. The cytoplasm contains mitochondria, ribosomes (Ri), endoplasmic reticulum (ER) and 
proacrosomal vesicles (pav). C, A zygotene primary spermatocyte contains an oval nucleus with dense 
heterochromatin and mitochondria in the cytoplasm. D, A pachytene primary spermatocyte (PSc) 
contains a nucleus with a bouquet pattern of heterochromatin. Note the presence of ER and mitochondria 
in the cytoplasm. 


(LSc, ZSc, PSc) are round, approximately 12-15 um in diameter, whereas the late-staged 
cells (DSc, MSc) are smaller in size (8-10 um in diameter). Other distinctive differences 
among various stages ofthe PrSc are the pattern of chromatin condensation and the relative 
amount of euchromatin versus heterochromatin. 


Male germ cells of abalone Molluscan Research 113 


Leptotene spermatocyte 


These cells are round, approximately 12-15 um in diameter (Fig. 14,3). The chromatin, 
which has started to condense into small blocks of heterochromatin, is scattered evenly 
throughout the nucleus. The nucleolus is still present, but not as prominent as that of the 
spermatogonium. The cytoplasm contains a few ribosomes, mitochondria and 
proacrosomal vesicles (Fig. 23). 


Zygotene spermatocyte 


These cells are approximately the same size as the LSc (approximately 12 um in 
diameter; Fig. 18). Under TEM, the heterochromatin blocks are denser than those of the 
LSc and the nucleolus disappears (Fig. 2C). The cytoplasm contains proacrosomal vesicles 
and mitochondria, similar to those in the LSc. 


Pachytene spermatocyte 


These cells are round and their sizes are smaller than the LSc (approximately 10—12 um 
in diameter; Fig. 1C). The heterochromatin appears as long threads and thick fibres that are 
entwined into a ‘bouquet pattern’, and are visible throughout the nucleus (Fig. 2D). The 
cytoplasm contains proacrosomal vesicles; the mitochondria and rough endoplasmic 
reticulum are greater in number than the LSc (Fig. 22). 


Diplotene spermatocyte 


These cells are similar to the PSc, but are smaller (approximately 8—10 um in diameter). 
The nucleus of the DSc is also smaller than that of the PSc. The chromatin strands, which 
are distributed among the denser nucleoplasm, become increasingly thicker than those in 
the earlier stage (Fig. 34). The cytoplasm contains mitochondria and proacrosomal 
vesicles, similar to those in the PSc. 


Metaphase spermatocyte 


These cells (approximately 8 um in diameter) exhibit thick chromosomes that are 
arranged close together along the equatorial region and the nuclear membrane completely 
disappears (Fig. 33). The cytoplasm contains mitochondria, numerous ribosomes and 
proacrosomal vesicles (Fig. 33). 


Secondary spermatocyte 


These cells are round and smaller than the MSc (approximately 6 um in diameter; 
Fig. 1C). They are rarely observed and occur after meiosis I. The SSc shows thick 
chromatin blocks composed of criss-crossing fibres, which appear as reticulate chromatin 
(Fig. 3C). Fewer mitochondria and proacrosomal vesicles are visible in the cytoplasm (Fig. 
3C). š 
Spermatids 
There are five stages of spermatid (St, ;), differing in size, nuclear shape and chromatin 
condensation pattern. 

Spermatid 1 


The nuclei of St, are round (approximately 6 um in diameter). The St, can be 
distinguished by their chromatin, which appears as homogeneous granules that are 


S. Singhakaew et al. 


114 Molluscan Research 


Fig. 3. 4, A diplotene primary spermatocyte (DSc) contains a round nucleus (Nu) vvith very thick 
heterochromatin (hc). Note the presence of mitochondria (Mi), ribosomes (Ri), proacrosomal vesicles 
(pav) and Golgi body in the cytoplasm. B, A metaphase spermatocyte (MSc) exhibits thick chromosomes 
arranged along the equatorial region. Mitochondria, ribosomes and proacrosomal vesicles are still present. 
C, A secondary spermatocyte (SSc) with a round nucleus showing reticulate chromatin (ch). D, Spermatid 
I (St,) showing a round nucleus with homogeneous granular chromatin. gc, Golgi complex. 


uniformly spaced throughout the nucleus (Fig. 3D). As a result, the whole nucleus appears 
moderately dense without any intervening transparent area of nucleoplasm. The cytoplasm 
exhibits fewer organelles, such as mitochondria, which tend to be concentrated on one side 


of the nucleus (Fig. 3D). 


Male germ cells of abalone Molluscan Research 115 


Spermatid II 


The general features of St, are similar to those of St,, but the nucleus, which is still 
round, is smaller in size in St; (approximately 5 um in diameter) and is located centrally 
within the cell (Fig. 44). The chromatin appears as homogeneous granules that are 
uniformly spaced throughout the nucleus and condensed into patches. The cytoplasm 
contains a few mitochondria on one side and ribosomes (Fig. 44). 


Spermatid III 


The St, is smaller than St; (approximately 4 um in diameter) and the nucleus maintains 
a round shape. The chromatin begins to condense into dark blocks, with intervening light 
areas of nucleoplasm (Fig. 4B). The cytoplasm contains few ribosomes, mitochondria and 
proacrosomal vesicles (Fig. 48). 


Spermatid IV 


The St, is smaller (approximately 3 um in diameter), but still appears round in shape. 
The chromatin of St, is almost completely condensed, resulting in an opaque nucleus 
(Fig. 4C). At this stage, there is a continued loss of cytoplasm, a decrease in nuclear size 
and condensation of nuclear material. The cytoplasm contains numerous ribosomes, a few 
large mitochondria and centrioles (Fig. 4C). 


Spermatid V 


The St; is the smallest among ther spermiogenic cells (approximately 2 um in diameter), 
but is still round in shape. The chromatin of St, is almost completely condensed (Fig. 4D). 
Fusion of proacrosomal vesicles into an acrosome that appears slightly indented at the 
anterior region of the nucleus can be seen (Fig. 4D). The cytoplasm contains numerous 
ribosomes and a few mitochondria (Fig. 4D). The mitochondria are fewer, but are larger in 
size, and occupy the posterior border of the nucleus (Fig. 4D). 


Spermatozoa 


Mature spermatozoa are detached from trabeculae and are arranged in rows further from 
the former stages, whereas their long tails are pointing outwards and are mingled with those 
of another spermatogenic unit (Fig. 12). The nucleus (1 x 4 um in size) is fully elongated 
and slightly tapered at the anterior end (Fig. 54). The chromatin is completely condensed 
and the anterior portion of the head is covered by an acrosome that appears as an inverted 
cup, the concavity of which separates it from the anterior border of the indented nucleus 
(Fig. 54). This subacrosomal space contains a crystalline acrosomal core embedded in 
more homogeneous material (Fig. 5C). The acrosomal matrix appears homogeneous, with 
varying degrees of electron opacity (Fig. 54). The nuclear chromatin is completely 
condensed (Fig. 5C). The head of each spermatozoon comprises a barrel-shaped nucleus 
(Fig. 54). At the posterior border of the nucleus, there are proximal and distal centrioles 
that are surrounded by a ring of five round mitochondria with cristae (Fig. 54.3). The tail, 
or flagellum, consists of a 9 + 2 arrangement of microtubules and is surrounded by a 
plasma membrane (Fig. 52). 


Discussion 


Most light microscopic studies on Haliotis have not categorised the various stages of 
spermatogenesis, apart from suggesting that there are four broad classes: spermatogonia, 


116 Molluscan Research S. Singhakaew et al. 


Fig. 4. 4, Spermatid II (St;) shows more condensed chromatin (ch) in a round nucleus (Nu). The 
cytoplasm exhibits fewer organelles. B, Spermatid III (St;) contains a round nucleus. Chromatin is 
condensed into dark blocks. Mitochondria (Mi) begin to form a cluster. C, Spermatid IV (St,) appears 
round in shape. The nucleus contains almost completely condensed chromatin. Mitochondria are located 
at the posterior border of the nucleus. Note the presence of centrioles (ce). D, Spermatid V (St;) contains 
a nucleus with almost completely condensed chromatin. Note the fusion of proacrosomal vesicles (pav) 
into an acrosome (Ac). Mitochondria are larger and occupy the posterior border of the nucleus. Ri, 
Ribosome. 


spermatocytes, spermatids and spermatozoa (Tomita 1967; Takashima ef al. 1978). 
Apisawetakan er al. (1997) studied the gametogenic processes in H. asinina using the light 
microscope and classified spermatogenesis into 13 stages: spermatogonium, five stages of 
primary spermatocyte, secondary spermatocyte, four stages of spermatid and spermatozoa 


Male germ cells of abalone Molluscan Research 117 


Fig. 5. Spermatozoa (Sz). A, Mature spermatozoa showing a fully elongated nucleus (Nu), acrosome 
(Ac) with subacrosomal space (S), proximal and distal centrioles (pc and dc, respectively) and a ring of 
mitochondria (Mi). B, High-power magnification of a ring of five mitochondria. C, High-power 
magnification of an acrosome. D, The tail (T) or flagellum consists of 9 2 arrangement of microtubules 
surrounded by a plasma membrane (pm). ce, Centriole. 


(immature and mature). Sobhon et al. (2001) reported 14 stages of spermatogenesis based 
on their ultrastructural study: spermatogonium, six stages of primary spermatocyte, 
secondary spermatocyte, four stages of spermatid and spermatozoa (immature and mature). 
The present study determined 13 stages of spermatogenesis in H. ovina: the 
spermatogonium, five stages of the primary spermatocyte, the secondary spermatocyte, 
five stages of the spermatid and the spermatozoa. We could not observe the diakinetic stage 
of the primary spermatocyte, only the metaphase spermatocyte. 


118 Molluscan Research S. Singhakaew et al. 


Like H. asinina (Sobhon et al. 1999, 2001), the spermatogonium of H. ovina is the 
earliest cell with a large nucleus containing a relatively large amount of euchromatin and a 
prominent nucleolus and little cytoplasm with free ribosomes and mitochondria. 
Spermatogonia divide mitotically to give rise to primary spermatocytes that pass through 
four stages of the first meiotic division. These prophase cells exhibit different forms of 
chromatin condensation. One remarkable characteristic of LSc, ZSc and PSc of both 
H. ovina and H. asinina is the presence of multiple proacrosomal vesicles in the cytoplasm. 
These vesicles begin to be synthesised in LSc and increase in PSc (Sobhon et al. 2001). In 
the MSc, the vesicles still appear quite numerous, whereas the chromatin becomes highly 
condensed into very large chromosomes. Thus, in this primitive gastropod, the 
proacrosomal vesicles are synthesised early in the LSc stage, even though they may be 
fused to form acrosomes much later in the late spermatid stages (Young and DeMartini 
1970; Hodgson and Bernard 1986; Healy ef al. 1998; Apisawetakan et al. 2000; 
Panasophonkul 2000; Sobhon er al. 2001). The fusion of multiple proacrosomal vesicles 
has been reported in several groups of vetigastropods, such as the trochids (Azevedo et al. 
1985; Hodgson ef al. 1990), bivalves (Hodgson and Bernard 1986) and scaphopods (Hou 
and Maxwell 1991). Although the very earliest stage of proacrosomal vesicle production 
was not observed in the present study, the ultimate source of these vesicles is undoubtedly 
the Golgi complex, as demonstrated previously for other vetigastropods (Azevedo et al. 
1985; Healy and Harasewych 1992) and bivalves (Hodgson and Bernard 1986). Secondary 
spermatocytes, similar to those of H. asinina (Sobhon et al. 2001), have heterochromatin 
that exhibits a checkerboard pattern. 

As mentioned earlier, there are five stages of spermatids in H. ovina, whereas Sobhon 
et al. (2001) described only four stages of spermatids in H. asinina. These classifications 
are based on size, chromatin granulation and condensation. Successive stages vary in size 
from 6 um in St, to 2 um in Stş. The major differences in the ultrastructure of developing 
spermatids of H. ovina and H. asinina are: (1) the formation of the acrosome begins in St, 
and is completed in St, for H. asinina (Sobhon et al. 2001), whereas this occurs at a later 
stage (St;) in H. ovina; (2) all stages of spermatids in H. ovina retain a round shape, whereas 
they vary from round and oval (St, s) to ellipsoid (St,) in H. asinina (Sobhon er al. 2001); 
(3) the acrosome of H. ovina appears slightly indented at the anterior region of the nucleus, 
whereas in H. asinina the acrosome only touches the nuclear envelope at the anterior end 
of the nucleus (Sobhon et al. 2001); and (4) the St, of H. asinina appears to be in a more 
advanced stage than that of H. ovina (i.e. there is a concentration of mitochondria in the 
posterior border of the nucleus and the centriole starts to form the axonemal complex of the 
tail (Sobhon er al. 2001); in H. ovina, only a concentration of mitochondria was found). 
There are two stages of spermatozoa (Sz, 5) in H. asinina (Apisawetakan et al. 2000; 
Sobhon ef al. 2001), whereas there is only one stage of spermatozoa in H. ovina. The Sz, 
is an immature spermatozoon with an acrosome and a short, developing tail, whereas Sz, 
is a mature spermatozoon. i 

The results of the present study show that the spermatozoa of H. ovina are very similar 
to those of H. asinina, except for the morphology of the sperm head. Haliotis ovina 
spermatozoa have a round to barrel-shaped head, whereas those of H. asinina are ellipsoid 
(Apisawetakan et al. 2000). In general, examination, to date, of spermatozoa in haliotid 
species shows that there are three types of sperm head: (/) short and globular or 
barrel-shaped (H. ovina; the present study); (2) ellipsoid (H. laevigata (Healy et al. 1998), 
H. diversicolor supertexta (Gwo et al. 1997) and H. aquatilis (Shiroya and Sakai 1992)); 
and (3) long and bullet-shaped (H. discus (Sakai et al. 1982; Usui, 1987), H. rufescens 


Male germ cells of abalone Molluscan Research 119 


(Lewis et al. 1980) and H. midae (Hodgson and Foster 1992)). Consequently, the sperm 
nuclei can also be classified into three types: round, ellipsoid and elongated. The chromatin 
of H. ovina, similar to that of H. asinina (Apisawetakan et al. 2000; Sobhon et al. 2001), 
appears to be of a granular type. This granular pattern of chromatin condensation can also 
be observed in other primitive gastropods, such as trochids (Hodgson ef al. 1990; Healy 
1996), scaphopods (Dufresne-Dube et al. 1993) and bivalves (Bozzo et al. 1993; Casas and 
Subirana 1994; Johnson et al. 1996). In H. ovina sperm, the acrosome is situated at the apex 
of the nucleus, as in H. asinina and also in many bivalves (Kubo 1977; Kubo and Ishikawa 
1978; Apisawetakan et al. 2000; Sobhon et al. 2001). 

The acrosome of H. ovina has an inverted cup shape, similar to those of H. asinina, 
H. laevigata, H. diversicolor supertexta and H. aquatilis (Gwo et al. 1997; Healy et al. 
1998; Apisawetakan et al. 2000; Shiroya and Sakai 1992). In contrast, the acrosomes of 
H. discus, H. rufescens and H. midae are much longer and narrower (Lewis et al. 1980; 
Sakai et al. 1982; Hodgson and Foster 1992). The acrosomal core consists of a 
crystalline-like axis embedded within a moderately dense matrix that occupies the whole 
subacrosomal space. The core is much shorter. The crystalline material probably consists 
of actin filaments and associated proteins, as reported in other molluscs (Baccetti and 
Afzelius 1976; Shiroya et al. 1986; Tilney et al. 1987; Healy 1989). This acrosomal core 
may participate in the extension of the acrosomal process during the acrosomal reaction and 
fertilisation (Apisawetakan et al. 2000). 

The tail ofa H. ovina sperm consists of five globular mitochondria located at the posterior 
end of the nucleus surrounding a pair of centrioles. A long axoneme stretches backwards 
from the distal centriole, which is surrounded by mitochondria. The axoneme core consists 
of 9 + 2 doublets of microtubules surrounded by a plasma membrane. This type of tail and 
midpiece was also observed in H. asinina (Apisawetakan et al. 2000; Sobhon et al. 2001), 
H. laevigata (Healy et al. 1998), H. aquatilis (Shiroya and Sakai 1992), H. diversicolor 
supertexta (Gwo et al. 1997) and also in several other vetigastropods (Azevedo et al. 1985; 
Healy 1989; Hodgson et al. 1990; Healy and Harasewych 1992) and in many bivalves 
(Hodgson and Bernard 1990; Bozzo et al. 1993; Casas and Subirana 1994; Healy 1996; 
Johnson et al. 1996), all of which reproduce by external fertilisation. It is apparent that the 
sperm of those molluscs with external fertilisation do not require larger quantities of energy 
than those of molluscs with internal fertilisation. Such sperm usually have midpieces that 
contain large cylindrical or helical mitochondria (Jaramillo et al. 1986; Gallardo and Garrido 
1989; Amor and Durfort 1990; Sretarugsa et al. 1991; Caceres et al. 1994). 

Gwo et al. (1997) suggested that large species of Haliotidae usually possessed long 
sperm heads and small species contain short sperm heads. If this is the case, then H. ovina 
sperm should be classified into the latter group. Sperm of H. ovina bear certain similarities 
to those of H. asinina, except for the shape of the sperm head. Further studies need to be 
performed on the ultrastructure of haliotid sperm. We anticipate that continued comparative 
work in this field will help to shed further light not only on species relationships within the 
Haliotidae, but also on the validity of the many proposed subgenera. 


Acknowledgment 
This investigation was supported by the Thailand Research Fund BRG/04/2543. 


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Tookvinart, S., Leknim, W., Donyadol, Y., Preeda-Lampabutra, Y., and Perngmak, P. (1986). “Survey on 
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www.publish.csiro.au/journals/mr Molluscan Research, 2003, 23, 123-148 


Gastropod phylogeny based on six segments from four genes 
representing coding or non-coding and mitochondrial or nuclear DNA 


D. J. Colgan^?, W E Ponder^, E. Beacham^ and J. M. Macaranas^ 


^The Australian Museum, 6 College Street, Sydney, NSW 2010, Australia. 
BTo whom correspondence should be addressed. Email: donc@austmus.gov.au 


Abstract 


Significant differences remain between gastropod phylogenetic hypotheses based on morphological and 
molecular datasets. We collected additional data from three gene segments (28S rDNA expansion region 
DI (36 taxa plus two from GenBank), cytochrome c oxidase subunit 1 (35 species plus one from Genbank) 
and small nuclear RNA U2 (24 species)). These were combined with data available for the same species for 
histone H3 and two other segments of 28S rDNA. Analyses of these data using cladistic, maximum 
likelihood or Bayesian methodologies were conducted in an attempt to resolve some of the differences 
between current hypotheses of gastropod relationships based on morphological and molecular data. The 
results were of particular interest in four areas. (7) Patellogastropoda in most analyses are included in a 
derived clade with some Vetigastropoda. In an analysis with Nautilus as the sole outgroup, transversions 
weighted threefold as costly as transitions and, with third codon position data ignored, Patellogastropoda 
are excluded from an otherwise monophyletic Gastropoda. (2) Cocculiniformia was never monophyletic in 
our analyses, although this possibility is not statistically rejected. (3) Nerita, the only representative of 
Neritopsina in this dataset, is placed anomalously in most analyses, but is, in a few cases, shown as a 
sister-group to the Apogastropoda, in accord with some morphological hypotheses. (4) Heterobranchia is 
rarely monophyletic in our analyses owing to the variable placement of the architectonicoid Philippea. This 
genus, even judged by the high levels of divergence within Heterobranchia, has undergone extreme rates of 
substitution. The Euthyneura is invariably monophyletic and nearly always included in a clade with the 
valvatoidean Cornirostra as its sister-group. 


Additional keywords: DNA sequence, heterobranch, multiple gene, Neritopsina, patellogastropod. 


Introduction 


Recent phylogenetic investigations of gastropods have used a variety of different datasets, 
ranging from shell morphology (including fossils, protoconch morphology and shell 
structure; Bandel 1997; Fryda, 1999; Wagner 2002; for a review, see Wagner 2001), 
ultrastructure (Ponder and Lindberg 1997; Künz and Haszprunar 2001), development 
(Freeman and Lundelius 1992; van den Bigelaar 1996; van den Bigelaar and Haszprunar 
1996; Lindberg and Guralnick 2001), pallial cavity structures (Haszprunar 1988a; Ponder 
and Lindberg 1996, 1997; Lindberg and Ponder 2001), overall morphology (Haszprunar 
1988a; Ponder and Lindberg 1996, 1997; Barker 2001), mitochondrial gene order 
(Kurabayashi and Ueshima 2000a, 20005) and molecular sequences. 

These studies show general agreement for the recognition of five major groups within 
Recent gastropods: (7) the Patellogastropoda (or Docoglossa), being the true limpets; (2) 
the Vetigastropoda (trochids, haliotids, fissurellids etc.); (3) the Neritopsina (or 
Neritimorpha), the nerites and relatives; (4) the Caenogastropoda (most of the 
“mesogastropods”, including the architaenioglossan taxa, and the neogastropods); and (5) 
the Heterobranchia (or Heterostropha as used by some palaeontologists (Bandel 1997), 


© Malacological Society of Australasia 2003 10.107 1/MR03002 1323-5818/03/020123 


124 Molluscan Research D. J. Colgan et al. 


whereas others (Ponder and Warén 1988) use Heterostropha as a paraphyletic subgroup 
within the Heterobranchia). The Caenogastropoda and Heterobranchia form the 
Apogastropoda in Ponder and Lindberg's (1997) sense. This taxon was originally 
introduced by Salvini-Plawen and Haszprunar (1987) to include the caenogastropods and 
only the basal heterobranchs, or “allogastropods”, in their usage, making it paraphyletic. An 
additional extant group, the Cocculiniformia (for a review, see Haszprunar 1998), is 
sometimes recognised and placed near the base of the gastropods. 

The first three groups and the Cocculiniformia comprise the paraphyletic 
'archaeogastropods', whereas the first four groups comprise the paraphyletic 
“prosobranchs”. The Heterobranchia, as first recognised by Haszprunar (1985a), contains 
the euthyneurans, a grouping of two taxa previously given subclass rank (the 
opisthobranchs and the pulmonates), as well as a number of assumed more “primitive” 
members, all of which were included within the “prosobranchs” at some time. These latter 
groups comprise the paraphyletic *Heterostropha' (sensu Ponder and Warén 1988) or 
“Allogastropoda” (Haszprunar 19855; Bandel 1997). 

Hickman (1988) advocated restricting the usage of the name Archaeogastropoda to the 
Vetigastropoda, whereas some (Bandel 1982, 1997; Bandel and Geldmacher 1996) 
formally use the same name for a group encompassing the patellogastropods together with 
the vetigastropods and the Cocculiniformia (i.e. excluding the Neritopsina), based 
primarily on their possession of a similar larval shell. However, the artificial nature of such 
a grouping is recognised (Fryda 1999). In stark contrast with this view is the idea that the 
patellogastropods represent the sister-taxon to the rest of the gastropods, a result suggested 
by most recent morphological studies (Golikov and Starobogatov 1975; Graham 1985; 
Haszprunar 1988a; Ponder and Lindberg 1996, 1997; Sasaki 1998; Barker 2001). It was on 
this basis that Ponder and Lindberg (1997) introduced the Eogastropoda, to encompass the 
Patellogastropods and their assumed coiled ancestors, and Orthogastropoda for the 
remainder of the gastropods. 

Gastropods date from the early Cambrian, although there is considerable speculation 
over which of the small univalve fossils in that period represent torted gastropods or 
monoplacophorans. For example, Parkhaev (2001) assigns Helcionelloidea s.s. to 
Gastropoda, whereas members of this group are often considered to be monoplacophorans 
(Wen 1990; Peel 1991). j 

Only recently have individual molecular investigations of gastropod phylogeny 
included examples from nearly all critical taxa in the class (McArthur and Koop 1999; 
Colgan et al. 2000). The genes now available for a broadly representative set of taxa 
include histone H3 (Colgan et al. 2000) and four regions of the 28S rDNA gene. These 
are the 27 (Tillier et al. 1992, 1994; McArthur and Koop 1999), Dó (Rosenberg et al. 
1997; McArthur and Koop 1999) and D4—5 (“28SA” in Colgan et al. 2000) expansion 
regions and a section near the D7 area including a new expansion region (“28SB” in 
Colgan ef al. 2000). With some notable exceptions, major groups are supported or weakly 
contradicted in molecular investigations. Nevertheless, the most comprehensive analysis 
(Colgan et al. 2000) is based on relatively short sequences (less than 900 aligned base 
positions), so it is not surprising that there are still disagreements between molecular and 
morphological (Haszprunar 1988a; Ponder and Lindberg 1997) assessments of gastropod 
relationships (Fig. 1). The collection of more molecular and morphological data offers the 
best chance of resolving such disagreements, although data from fossils are also being 
examined rigorously and may well assist in further elucidating gastropod phylogeny 
(Wagner 2001). 


Gastropod molecular phylogenetics Molluscan Research 125 


Ischnochiton Polyplacophora 
Trichomya 
Anadara Bivalvia | 
Nautilus Cephalopoda 
Cellana 
Eogastropoda Notoacmaea Patellogastropoda 
Montfortula 
Perotrochus 
Austrocochlea 
Lepetodrilus 
Notocrater Pseudococculinidae 
Depressigyra Peltospiridae 
B Leptopoma j " 
E Bellamya Architaenioglossa | 
Campanile 
Nodilittorina 
d Serpulorbis 
Strombus 
Cypraea 
Cabestana 
Ataxocerithium 
Epitonium 
Nassarius 
Dicathais 
b Mitra Neogastropoda 
Conus 
Cancellaria 
Zeacumantus 
d PEN Heterostropha] 
Onchidium 
Salinator 
Hedleyoconcha 
Siphonaria 
Ophicardelus 
Aplysia 4 = 
Bullina Ophisthobranchia 
Coccopigya Cocculinoidea 
Nerita Neritopsina 


Vetigastropoda 


Orthogastropoda 
Apogastropoda 
Sorbeoconcha 


g 
o 
o 
o. 
o 
Iz] 
= 
o 
g 
D 
o 
c 
o 
g 
o 


Pulmonata 


Heterobranchia 
Euthyneura 


Fig. 1. Gastropod phylogeny based on the morphological analyses of Ponder and Lindberg (1997). 
Families not included in their analyses are-placed according to their general taxonomic classification (i.e. 
Cancellariidae and Mitridae in Neogastropoda, Onchidiidae, Siphonariidae, Ellobiidae and Charopidae 
in the Pulmonata). Taxa studied by Ponder and Lindberg (1997) but not here are pruned from the tree. 
Names of higher categories follow Ponder and Lindberg (1997). Differences of this topology from the 
Haszprunar (1988) topology are indicated. An upper case letter on a clade in the Ponder and Lindberg 
(1997) tree indicates that it is shown at the point specified by the corresponding lower case letter in the 
Haszprunar tree. 


In the present paper, we report analyses of an enlarged molecular dataset principally 
addressed to four areas of disagreement with morphologically based hypotheses, namely: 
(7) the position of the Patellogastropoda; (2) the monophyly and relationships of 
Cocculiniformia; (3) the relationships of Neritopsina; and (4) the monophyly of the 
Heterobranchia. Parts of two genes not previously used in overall gastropod phylogeny 
(cytochrome c oxidase subunit 1 (CO/) and small nuclear U2 RNA (snU2 RNA)) have been 
sequenced and new sequences from the 27 28S rDNA expansion region have been collected 
for most species studied in Colgan et al. (2000). The new genes.extend the range of gene. 
types used in gastropod phylogeny because they are respectively mitochondrial coding 
(CO1) and nuclear non-coding (snU2 RNA). The snU2 RNA is a component of the 


126 Molluscan Research D. J. Colgan et al. 


spliceosome (Guthrie and Patterson 1988) that has previously provided useful characters 
for higher-level phylogenetic studies of Arthropoda (Colgan et al. 1998) and Polychaeta 
(Brown et al. 1999). 

The division of Gastropoda into Eogastropoda and Orthogastropoda has not been 
supported in comprehensive molecular studies to date. In Colgan et al. (2000), 
Patellogastropoda plus a cocculiniform limpet (Cocculinoidea: Coccopigya) was a 
sister-group to the remainder of the studied species. A similar topology is seen in analyses 
of the approximately 450 (aligned) base pairs of /8S rDNA in the compiled dataset of 
Harasewych and McArthur (2000), where Patellogastropoda plus a monophyletic 
Cocculiniformia is a sister-group to all other gastropods. In McArthur and Koop (1999), 
where Cocculiformia are not represented, Patellogastropoda is a sister-group to 
Apogastropoda. Statistically, no placement of Patellogastropoda as a sister-group to another 
major clade has a significantly higher likelihood than any other in the McArthur and Koop 
(1999) analyses. 

Monophyly of 'cocculiniform' limpets is one of the major areas of disagreement 
between the Haszprunar (19885) and Ponder and Lindberg (1996, 1997) morphological 
topologies. The two main constituent groups, Cocculinoidea and Lepetelloidea, comprise a 
monophyletic Cocculiniformia (Haszprunar 1988a; 1998) or are diphyletic (Ponder and 
Lindberg 1996, 1997). The molecular datasets also disagree. Cocculiniformia are 
diphyletic in Colgan er al.(2000), Notocrater houbricki (Lepetelloidea) being placed in 
Vetigastropoda and Coccopigya hispida (Cocculinoidea) with Neritopsina in accordance 
with Ponder and Lindberg (1997). Cocculiniformia represented by Cocculina messingi 
(Cocculinoidea) and N. houbricki are monophyletic (albeit with weak support) in 
Harasewych and McArthur (2000), using partial 78S rDNA data, and are a sister-group to 
Patellogastropoda. 

The relationship of the Neritopsina (or Neritimorpha) to the rest of the gastropods is 
unresolved with two main scenarios: they are either a sister-group to the vetigastropods and 
the apogastropods or a sister-group to the apogastropods. Fryda (1999), Bandel and Fryda 
(1999) and Bandel (2000) have discussed the fossil evidence for the evolution of this group 
since its first undoubted appearance in the Late Silurian-Devonian (428—374 million years 
ago; Fryda and Blodgett 2001), although earlier origins have been argued. 

Within the Heterobranchia, Euthyneura, comprising two of the three classes 
(Opisthobranchia and Pulmonata) in Thiele’s (1925, 1929-31) classification, are 
monophyletic in most recent molecular analyses where few taxa are used, but not in studies 
with larger taxonomic samples (Thollesson 1999; Dayrat et al. 2001). In morphological 
analyses involving Recent taxa, Heterobranchia is the sister-clade to Caenogastropoda 
(Haszprunar 1988a; Ponder and Lindberg 1996, 1997). In McArthur and Koop (1999), the 
only included heterostrophan (Valvata sp.) is a sister-taxon to Euthyneura. In Colgan et al. 
(2000), two representatives of the group are included. They are monophyletic but not a 
sister-group to Euthyneura, possibly owing to the high degree of autapomorphy in their 
sequences, this being particularly notable for the architectonicoid species Philippea lutea 
(see below). 


Materials and methods 
Materials and molecular methods 


The taxa used, collection and registration data of specimens, methods and tissue types used for DNA 
extraction are given in Colgan et al. (2000). Our naming of higher groups follows Ponder and Lindberg 
(1997). Specimen voucher numbers are given in Table 1. 


Gastropod molecular phylogenetics Molluscan Research 127 


Primer pairs for U2 are given in Colgan et al. (1998). The primers for CO7 were as follows: Cox AF, 
CWAATCAYAAAGATATTGGAAC (41); Cox AR, AATATAVVACTTCVVGGGTGACC (725); and Cox 
623R, GGTAARTYTATTGTAATAGCWCC (623). The figures in parentheses refer to the 3’ end of the 
oligonucleotide in the sequence of Lumbricus terrestris (Boore and Brown 1995; GenBank accession 
LTU24570). Primers Cox AF and AR were used together to produce a 690 bp product. Where a product was 
not obtained using this pair, Cox 623R was used with Cox AF to give a 626 bp product. 

The primers for the D/ expansion region were as follows: 288 DIF, ACCCSCTGAAYTTAAGCAT 
(43); 28S DIR, AACTCTCTCMTTCARAGTTC (406). Figures in parentheses refer to the 3 end of the 
oligonucleotide in the mouse 28S rDNA sequence (X00525; Hassouna ef al. 1984). Primer DIF was taken 
from Macarthur and Koop (1999) and DIR was designed from an alignment of Ascaris lumbricoides 
(U94751), Drosophila melanogaster (M21017, M29800) and Mus musculus (X00525) sequences. 

The basic polymerase chain reaction (PCR) profile was as follows: 959C for 5 min, 43—549C for 45 s, 
72°C for 1 min for one cycle; 95°C for 30 s, 43—54?C for 45 s, 72°C for 1 min for 30-34 cycles; and 95°C 
for 30 s, 45—60°C for 45 s, 72°C for 5 min for the final cycle. The annealing temperatures varied according 
to the primers’ specificity for the different taxa and were usually 50-52°C for U2, 52-54°C for D1 28S 
rDNA and 43-45?C for CO1. To obtain PCR products from difficult samples, 20 uL GeneReleaser™ 
(Bioventures, Murfreesboro, TN, USA) was added to the DNA template and microwaved for 6 min. The 
remaining PCR mix was immediately added and cycling commenced. Reaction products were resolved on 
2% agarose gels containing ethidium bromide. All single band products were purified using the 
QIA quickTM PCR Purification Kit (Qiagen, Venlo, The Netherlands) and, where multiple band products 
were obtained, the correct sized band was excised from 2% low-melt agarose in TAE buffer (0.04 M Tris , 
0.001 M EDTA (pH 8.0), 0.02 M glacial acetic acid) and purified using the same kit. Products were 
sequenced in both directions by the Applied Biosystems (ABI*; Norwalk, CT, USA)310 DNA Sequencing 
System using the DyeDeoxy™ Terminator sequencing method (Big Dye?" version | or 2; ABI), according 
to the manufacturer's instructions. The consensus sequence for each individual was obtained by reconciling 
forward and reverse sequences using Sequence Navigator (ABI). 

The GenBank accession numbers of sequences used in Colgan et al. (2000) are AF033716-AF033794 
for 2584 rRNA and 2888 rRNA and AF033675-AF033715 for H3. Nautilus pompilius CO1 data are from 
AF000054 (Carlini and Graves 1999). Sequences for Viviparidae (U75863) and Cornirostridae (U75862) 
were obtained from GenBank (McArthur and Koop 1999). The GenBank accession numbers for the new 
sequences are AY296815—AY296850 for CO1, AY296873—AY296909 for zə DI and AY296851— 
AY296872 for snU2 RNA. 


Phylogenetic analysis 


Sequences were aligned using the default values in CLUSTAL W (Thompson ef al. 1994). The CLUSTAL 
output was inspected and, where required, indels were edited by hand. The CO/ and U2 sequences required 
no modification, but some regions of the D/ segment were. altered. These were specified as regions of 
uncertain alignment and were not used for analyses. All bases in the 2854 and H3 alignments from Colgan 
et al. (2000) were used. The region of uncertain alignment in 28S rDNA B in Colgan er al. (2000) was not 
used. The alignments are available from the authors and as Accessory Material from the journal's website. 

PAUP* 4.0b10 (Swofford 2000) was used for maximum parsimony analysis by conducting heuristic 
searches (100 iterations with random input order) with the default options (unless otherwise stated below). 
All characters were assumed unordered and indels treated as unknown in all reported analyses. One hundred 
bootstrap pseudoreplicates (Felsenstein 1985) were conducted with 20 random input replicates at each 
replicate to estimate support for nodes. Bremer decay indices were calculated using AutoDecay version 
3.03 (Eriksson and Wikstróm 1996) 

Maximum likelihood analyses of a reduced taxon set were performed using 10 random addition 
sequences for heuristic searches with the following settings. The number of substitution types was two, with 
the transition/transversion ratio estimated by maximum likelihood. Empirical nucleotide frequencies were 
assumed. The assumed proportion of invariable sites was zero, with a gamma discrete approximation (with 
shape parameter 0.5) using four rate categories. Another ML analysis was conducted with the same 
assumptions except that the maximum parsimony trees were used to start the analysis, branch swapping was 
by subtree pruning and reconnection, and five replicates were used. Quartet puzzling (100 000 steps) was 
performed using PAUP with the same likelihood settings, entering the transition/transversion ratio 
estimated during the likelihood searches by hand. 

A Bayesian analysis was conducted with the program MrBayes (Huelsenbeck and Ronquist 2001) using 
the same likelihood parameters as the maximum likelihood analysis and sampling a tree every 100 steps 


. J. Colgan et dl. 


Molluscan Research 


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130 Molluscan Research D. J. Colgan et al. 


along a 100 000 step Markov chain. Only two simultaneous chains were run, owing to computer memory 
restrictions, and the first 43 300 steps were discarded because convergence to an area of stable likelihood 
did not occur until this time. 

All analyses listed below, except (ix)-(xi), were conducted with maximum parsimony. 


(i All data, excluding areas of uncertain alignment in the 285 rDNA. 

(ii) All data, excluding areas of uncertain alignment in the 28S rDNA and third codon positions. 

(iii) All data, excluding areas of uncertain alignment in the 28S rDNA, transversions weighted threefold 
as costly as transitions. 

(iv) All data, excluding areas of uncertain alignment in the 28S rDNA and third position data, 
transversions weighted threefold as costly as transitions. 

(v) All data, excluding areas of uncertain alignment in the 28S rDNA, transversions weighted threefold 
as costly as transitions; Nautilus was used as the only outgroup. 

(vi) All data, excluding areas of uncertain alignment in the 28S rDNA and third codon position data, 
transversions weighted threefold as costly as transitions; Nautilus was used as the only outgroup. 

(vii) All data, excluding areas of uncertain alignment in the 28S rDNA, and excluding Philippea lutea. 

(viii) All data, excluding areas of uncertain alignment in the 28S rDNA and third codon position data, and 
excluding Philippea lutea. 

(ix) A maximum likelihood analysis of a reduced dataset using random taxon addition and excluding 
areas of uncertain alignment in the 28S rDNA. 

(x) A maximum likelihood analysis using maximum parsimony starting trees excluding areas of 
uncertain alignment in the 285 rDNA. 

(xi) A Bayesian analysis of all data excluding areas of uncertain alignment. 


Although analyses of individual genes are not reported in detail, they were conducted to confirm that 
there was no cross-contamination between sequences within this study or the inclusion of sequences from 
other material treated in this laboratory. 

MacCLADE (Maddison and Maddison 1992) was used to set character types and substitution weights 
and to compare trees with the *winning-sites test” (Prager and Wilson 1988) using the “compare two tree 
files’ option. Files containing all trees from each analysis were used. One tree (or set of trees) was preferred 
to another if the number of characters for which it had less steps than the alternative tree(s) was significantly 
greater than the number of such characters for the alternative. Probabilities were estimated using a 
two-tailed normal approximation. 


Results 


The number of aligned bases excluding the areas of uncertain alignment, the number of 
variable characters and the number of parsimony informative characters (in order for the 
individual gene segments) were: D] 28S rDNA, 317, 154 and 113 respectively; 2854, 255, 
111 and 71 respectively; 28SB, 256, 73 and 47 respectively; CO1, 567, 400 and 318 
respectively; histone H3 274, 121 and 107 respectively; and snU2 RNA 131, 56 and 41 
respectively. J 
The mean GC content of the studied species for s U2 RNA was lower (46.11%) than the 
other non-coding RNAs and notable differences were observed between the three 28S 
rDNA segments: 57.35% for the D/ region, 50.67% for 28SA and 48.67% for the 2888 re- 
gion. The GC content was 58.42% for histone H3. The percentage of GC in CO1 was very 
low (38.36%), but increased to 45.37% with the exclusion of third-position bases, consist- 
ent with the usual pattern for animal mitochondrial coding DNA (Wirth et al. 1999). 
Chi-square tests suggest that the percentage nucleotide composition was homogeneous 
within the studied species for H3 (P = 0.571), U2 (P = 1) and all 28S segments (P = 1 for 
D1 28S rDNA, 2834 and 28SB), but was significantly inhomogeneous for CO! (P < 0.001). 
This inhomogeneity was due to third codon position data; when these data were omitted, the 
hypothesis of compositional homogeneity was not rejected (P = 0.999). 

The average transition to transversion ratios for the six gene segments based on the 
Kimura two-parameter distance, and ignoring the areas of uncertain alignment and pairwise 


Gastropod molecular phylogenetics Molluscan Research 131 


comparisons without transversions, were: 1.792 + 1.560 for U2 (range range 0-13.31); 
1.260 + 0.600 for H3 (range 0.348—5.260); 1.276 + 0.874 for DI 28S rDNA (range 
0—12.036); 3.017 + 2.790 for 28SA (range 0—26.065); 2.555 + 3.602 for 28SB (range 
0-18.622); and 1.063 + 0.306 for CO1 (range 0.456—-2.345). Ratios were also examined 
omitting the third position of H3 and COI. For H3, the average was 1.980 + 1.028 (range 
0.279—9.793). For CO1, the average was 1.372 + 1.376 (range 0.279—21.186). 

Incongruence length difference analysis for the data set for analysis (7) with 100 
replicates returned a probability of 0.27 that the phylogenetic implications of the various 
gene segments do not differ. For other analyses, the probabilities are: (ii) 0.99; (iii) 0.99; 
(iv) 0.01; (v) 0.99; (vi) 0.99; (vii) 0.01; and (viii) 0.62. Summaries of maximum parsimony 
analyses for individual genes are given in Table 2. Generally, few clades receive bootstrap 
support greater than 50% in these analyses, so the results are not discussed in detail. 

Details of the various analyses, including the number of maximum parsimony trees, the 
consistency index, the length of the trees and the minimum length of trees satisfying the 
Ponder and Lindberg (1997) topology, are presented in Table 3. The maximum parsimony 
trees for analyses (1), (ii), (iii), (vi) and (ix) are presented in Figs 2-6. The results for other 
analyses are compared with these figures below. Their bootstrap supported clades are 
indicated in Table 2. 

Figure 2 shows one of two maximum parsimony trees for analysis (i). The topology of 
the second differs only above point A on the figure (with bolded branches found in both 
trees). The topology with Philippea removed (analysis (vii)) is the same as for analysis (i) 
(omitting Philippea) at nodes below the arrows on Fig. 2. The topologies differ above the 
arrowheads, notably in that Opisthobranchia and Pulmonata are monophyletic sister-groups 
in analysis (vii). Bootstrap supported clades are the same as for analysis (7) and have similar 
percentages. 

The topology for analysis (viii) is the same as for analysis (ii) (with the removal of 
Philippea) at nodes below the arrow on Fig. 3, except that Patellogastropoda is a 
sister-group to the clade comprising (Montfortula, Austrocochlea, Notocrater and 
Lepetodrilus). Branches above the arrow seen in the strict consensus for analysis (ii) and 
for analysis (viii) are indicated in bold. 

Figure 4 shows one of the two maximum parsimony trees for analysis (iii). The second 
tree differed in the placement of some taxa in the area between Nodilittorina and Bellamya, 
with Nerita shown in the equivalent position to Bellamya in the first tree. In analysis (v) 
Nautilus, Coccopigya and the other gastropods form a basal trichotomy. The gastropods are 
then divided into: (a) Euthyneura; and (b) the remaining taxa. Group (b) is further divided: 
(b.1) Leptopoma and Campanile; and (b.2) other Caenogastropoda, Vetigastropoda, 
Patellogastropoda and Philippea. 

The topology for analysis (iv) is similar to that for analysis (vi). Addition of the other 
outgroup taxa in analysis (iv) places the root of the tree at the position marked by A in 
Fig. 5. 

The maximum likelihood topology of the reduced taxon set is shown in Fig. 6. The 
estimated transition/transversion ratio on which this is based was 1.64. The estimated ratio 
for analysis (x) was 1.62. Analysis (x) produced two trees differing only in some placements 
within Caenogastropoda. The primary dichotomy lay between a group comprising 
Patellogastropoda, Notocrater and the Vetigastropoda except Perotrochus and the other 
gastropods. Caenogastropoda was monophyletic except that it anomalously included Nerita 
and that Leptopoma was grouped with Nautilus plus Philippea as a sister-group to a clade 
comprising Coccopigya, Pleurotomaria and Depressigyra. Euthyneura was monophyletic 


Molluscan Research . J. Colgan et al. 


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134 Molluscan Research D. J. Colgan et al. 


Ischnochiton 


100 Trichomya SO cnanges 
15 Anadara 
Nautilus 

3 Philippea Heterostropha - part 

Leptopoma Architaenioglossa - part 

4 56 Perotrochus Vetigastropoda - part 

7 Depressigyra Peltospiridae 

5 Coccopigya Cocculinoidea 

Nerita Neritidae 

3 Cypraea 
Zeacumantus t 
Bellamya Architaenioglossa — part | $ 
6 Campanile I 
4 Strombus S 
ç Y Cabestana ° 
3 Ataxocerithium o 
A baz Cancellaria* m 
4 Nodilittorina S 
3 Dicathais* o 
0 Serpulorbis o 
4 2 Epitonium ó 
Nassarius* 
Mitra* 
2 Conus* 

Cornirostra Heterostropha —part 

3 Onchidium S 
Hedleyoconcha 

97 zs Siphonaria Pulmonata E 
9 8 Ophicardelus £ 
Pog Salinator 3 
Bullina i C 

20 Aplysia Opisthobranchia 

100 Cellana 
62 Notoacmaea Patellogastropoda 
3 Notocrater Pseudococculinidae 
3 Montfortula 
2 Austrocochlea ba Vetigastropoda- part 


3 Lepetodrilus 


Fig. 2. One of two maximum parsimony trees for all data, excluding areas of uncertain alignment 
(analysis (i)). The topology of both is identical below point A. The arrowheads indicate topological 
similarities with analysis (vii) detailed in the text. Bold branches above A are also seen in the strict 
consensus of analysis (i) and analysis (vii). Bootstrap percentages above 50% are shown above a branch. 
Bremer decay indices are shown below the branch. Branches without figures have indices of 1. Asterisks 
on genus names indicate membership of Neogastropoda. Note that the brackets on the right-hand side do 
not necessarily show monophyletic clades. 


with Cornirostra its sister-group. The clades with puzzling support more than 5096 were: 
(Nautilus, Philippea) 55%; ((Irichomya, Anadara) 8496; (Cellana, Notoacmaea) 5196; 
((Austrocochlea, Lepetodrilus) 68%; Montfortula) 61%; ((Perotrochus, Depressigyra) 
65%; Coccopigya) 56%; (Ataxocerithium, Cancellaria) 60%; Pulmonata 52%; 
Ophisthobranchia (here Aplysia, Bullina) 69%; (Euthyneura plus Cornirostra) 60%. 

The topology of the Bayesian analysis (xi) with /schnochiton as the outgroup is broadly 
similar to the results of other analyses, albeit with many instances of high “posterior 
probabilities" of clades that are unexpected on morphological grounds but that are shown 


Gastropod molecular phylogenetics Molluscan Research 135 


Ischnochiton 
Trichomya 
18 Anadara 
Nautilus 
Coccopigya Cocculinoidea 
Leptopoma Architaenioglossa — part 
Campanile 
Bellamya Architaenioglossa -part 
Nodilittorina 
60 Serpulorbis 
2 3 Epitonium 
2 Ataxocerithium 
4 Cypraea 
2 Zeacumantus 
0 Mitra* 
Dicathais* 
Conus* 
Nassarius* 
Strombus 
Cabestana 
Cancellaria* 
Cornirostra Heterostropha -part 
53 Onchidium 
Ophicardelus 
Salinator Pulmonata 
Hedleyoconcha 
14 Siphonaria 
Bullina Opisthobranchia 
100 — Cellana 
= Nice Patellogastropoda] 
2 0 Philippea Heterostropha - part 
Montfortula h 
92| 68 yos Vetigastropoda -part | 
a Notocrater Pseudococculinidae 
4 Lepetodrilus i E 
Perotrochus MOET TELE part] 
2 Depressigyra Peltospiridae 
Nerita Neritidae 


Caenogastropoda 


93 


Euthyneura 


Fig.3. The strict consensus tree for analysis (ii), all data excluding areas of uncertain alignment and third 
positions. Bootstrap percentages above 50% are shown above a branch. Bremer decay indices are shown 
below the branch. Branches without figures have indices of 1. Moving branch A to branch a makes the 
topology of this analysis identical to that for analysis (viii) at points below the arrowhead. All Euthyneura 
except Siphomaria are included in a clade with bootstrap support of 54%, although not found in the 
maximum parsimony tree. Asterisks on genus names indicate membership of Neogastropoda. Note that 
the brackets on the righ-hand side do not necessarily show monophyletic clades. 


in parsimony or maximum likelihood analyses without great support. For instance, the 
anomalous pairing of Nerita with Cypraea has a posterior probability of 0.99; the pairing 
of Strombus with Nassarius in a group including most Neogastropods has a probability of 
0.70 and the pairing of the remaining neogastropod Cancellaria with Ataxocerithium has a 
probability of 0.99. 

Patellogastropoda was monophyletic with high bootstrap support in all parsimony 
analyses, but was shown in a basal position only in analysis (vi) (Fig. 5). In other analyses, 
the group was included in a clade with some vetigastropods and Notocrater (analyses (i), 
(vii); Fig. 2), with this group plus Depressigyra (analysis (x)), paired with Philippea 
(analysis (iij) as a sister-group to a clade comprising other heterobranchs, 
caenogastropods, Coccopigya, Nerita and Nautilus (Fig. 4); or paired as sisters with 
Philippea (analyses (ii), (v); Fig. 3), Nautilus (analysis (iv)) or Notocrater, Montfortula, 


136 Molluscan Research D. J. Colgan et al. 


Ischnochiton 
100 Trichomya 
74 Anadara 
9 Depressigyra Peltospiridae 
9 Pleurotomaria 
9| 94 Montfortula i 
31 57 Austrocochlea ..... 
7 Lepetodrilus 
10 Notocrater Pseudococculinidae 
Nautilus dö 
12 Coccopigya Cocculinoidea 
Cornirostra Heterostropha- part 
Onchidium 
73 54] 8 Hedleyoconcha S 
22 3 Salinator Pulmonata - part ° 
2| 2 Ophicardelus > 
2 98 [3 əə Opisthobranchia = 
33 Sibhonaria Pulmonata — part | !!! 
Leptopoma Architaenioglossa - part 
2 Campanile t 
Nodilittorina ° 
Serpulorbis A 
4 Til? Epitonium U 
Nassarius* e 
9 Strombus 9 
4 12 Cabestana E. 
Ataxocerithium Oo 
2İ 2 Cancellaria” e 
A 2 Dicathais* ° 
5 Conus* o 
Zeacumantus 
7 7 Mitra” 
Nerita Neritidae 
12 Cypraea Caenogastropoda- part 
7 Bellamya Architaenioglossa — pan | 
100 
52 [7439 Not Apalan Patellogastropoda | 
14 Philippea Heterostropha — part 
— 100 changes 


Fig. 4. One of two maximum parsimony trees for all data, excluding areas of uncertain alignment with 
transition/transversion weighting (analysis (iii)). Bootstrap percentages above 50% are shown above a 
branch. Asterisks on genus names indicate membership of Neogastropoda. Note that the brackets on the 
right-hand side do not necessarily show monophyletic clades. 


Austrocochlea and Lepetodrilus (analysis (viii)) within a grouping of all vetigastropods and 
hot-vent taxa except Coccopigya. In no case did the pairing of Patellogastropoda as 
sister-groups with any other taxon receive bootstrap support greater than 50%. In analysis 
(xi), Patellogastropoda is a sister-group to the grouping of all vetigastropods and hot-vent 
taxa except Coccopigya with a posterior probability of 0.52. Imposing the constraint that 
Eogastropoda and Orthogastropoda were monophyletic sister-groups required 32 more 
steps (P ~ 0.11 using the winning-sites test). 


Gastropod molecular phylogenetics Molluscan Research 137 


Nautilus 
100 Cellana 
125 Notoacmaea 
Montfortula Vetigastropoda — part | 
94 Austrocochlea 
55| 26 Notocrater Pseudococculinidae 
3 Lepetodrilus Vetigastropoda -part 
Depressigyra Peltospiridae 
Perotrochus Vetigastropoda - part 
Coccopigya Cocculinoidea 
Cornirostra 
Philippea Heterostropha 
60 Onchidium 
Ophicardelus 
Hedleyoconcha 
= 13 Salinator 
Bullina = 
98 i 
13 Aplysia Opisthobranchia 
23 Siphonaria Pulmonata- part 


B Leptopoma Architaenioglossa 
Bellamya 


f Nodilittorina 
70 Serpulorbis 
5 LS3T 7 Epitonium 
3 Ataxocerithium 
Strombus 
Cypraea 
Cabestana 
Zeacumantus 
Nassarius* 
A => Dicathais* 
3 Mitra* 
Conus* 
Cancellaria* 
Campanile 
Nerita Neritidae 


Patellogastropoda 


Pulmonata- part 


Euthyneura 


Caenogastropoda 


Fig. 5. The strict consensus tree for analysis (vi), all data excluding areas of uncertain alignment and 
third positions, with Nautilus as the only outgroup and transition/transversion weighting. Bootstrap 
percentages above 50% are shown above `a branch. Bremer decay indices are shown below the branch. 
Branches without figures have indices of 1. “A” indicates the root when the other outgroup taxa are added 
(analysis (iv)). The other differences between the strict consensus trees of these two analyses is that branch 
B moves to b and branch C to c in analysis (iv). Asterisks on genus names indicate membership of 
Neogastropoda. Note that the brackets on the right-hand side do not necessarily show monophyletic 
clades. 


Cocculiniformia was never monophyletic in our analyses. Coccopigya was a 
sister-group to Euthyneura plus Cornirostra in analyses (ii), (iv), (vi), (viii), (ix), (x) and 
(xi), with significant bootstrap support in analyses (i7) and (viii) and a posterior probability 
of 100 in analysis (xi). It was a sister-group to Nautilus in analysis (iii) and Perotrochus plus 
Depressigyra in analysis (i) and analysis (vii) and formed one member of a basal trichotomy 
with Nautilus in analysis (v). Notocrater was always closely associated with a group of 
Vetigastropoda (Montfortula, Austrocochlea and Lepetodrilus). This group of four taxa was 
monophyletic with high support in all analyses except analyses (i) and (vii), where it also 
included the Patellogastropoda (as a sister-group to Notocrater). Even in analyses (i) and 
(vii), although the group of four was not shown in maximum parsimony trees, it received 


138 Molluscan Research D. J. Colgan et al. 


Ischnochiton 
62 Trichomya 
Philippea Heterostropha- part 
71 Cellana 

Notoacmaea Patellogastropoda 
Coccopigya S 
Cornirostra Heterostropha- part 3 
zu 81 Onchidium ° 
57 Hedleyoconcha Pulmonata | = 
Aplysia Opisthobranchia | 5 
| Nerita ut m 
Cypraea ” Neritidae œ 
Nodilittorina ° 
Ataxocerithium o. 
Nassarius” £ 
Conus* E 
Leptopoma ^ 5 5 
Bellamya Architaenioglossa | S 
Campanile S 

Montfortula Ç 

Austrocochlea Vetigastropoda- part | ° 

Notocrater Pseudococculinidae 

ule Perotrochus Vetigastropoda- part 

Depressigyra Peltospiridae 

Nautilus 


— 0.05 substitutions/site 


Fig. 6. Maximum likelihood tree of a reduced taxon set, including all data, excluding areas of uncertain 
alignment. Asterisks on species names indicate membership of Neogastropoda. The figures above 
branches indicate puzzling support of more than 5096: Nodilittorina plus Nassarius (5490), 
Ataxocerithium plus Conus (60%) and Montfortula plus Austrocochlea (64%) received puzzling support 
more than 50%, despite not appearing as clades in the likelihood tree. Note that the brackets on the 
right-hand side do not necessarily show monophyletic clades. 


bootstrap support of more than 60%. Relationships within the group varied, with 
Notocrater being found as a sister-group to each of the three other members in at least one 
analysis. Imposing the constraint that Cocculiniformia is monophyletic required 18 more 
steps (P = 0.10 using the winning-sites test). 

Euthyneura was monophyletic in all analyses with high bootstrap support (Figs 2-6; 
Table 2). Pulmonata and Opisthobranchia were both monophyletic only in analyses (vii), (x) 
and (xi), here with high posterior probabilities for each clade. In some other analyses (i and 
ii), Opisthobranchia was paraphyletic with respect to Pulmonata, but the groups were 
intermingled in analyses (iii), (iv), (v), (vi) and (vii), with bootstrap support for a clade of 
all Euthyneura except Siphonaria in analysis (iv) (8596) and analysis (vi) (80%). 

In all analyses, Cornirostra was closely associated with Euthyneura, being shown with 
high bootstrap support or posterior probability as the sister-group to this clade except for 
analyses (iv) and (vi). In these analyses, Cornirostra was paired with Philippea as a 
sister-group to Euthyneura to form a monophyletic Heterobranchia, with bootstrap support 
of 67% in analysis (iv). Generally, Heterobranchia was disrupted by the association (not 
bootstrap supported) of Philippea with other taxa: Nautilus in analyses (i) and (x); and 
Patellogastropoda in analyses (ii), (iii) and (v). Imposing the constraint that Heterobranchia 
is monophyletic required 23 more steps (P = 0.22 using the winning-sites test). 

The genetic divergence of the Heterobranchia as measured by the distance of terminals 
from the root in maximum parsimony analyses is striking, although less pronounced in 
likelihood analysis. 


Gastropod molecular phylogenetics Molluscan Research 139 


In each analysis except analyses (v) and (vii), the great majority of the Caenogastropoda 
and Heterobranchia grouped to form a recognisable but weakly supported *apogastropodan 
clade’. Examples of the exclusion of Philippea from this are listed above. Lepotopoma was 
excluded in analyses (i) and (vii). Unexpectedly included taxa are Nerita in analysis (i), (iv), 
(viii), (ix) and (x), Nautilus and Coccopigya in analyses (ii) and (iii), and Coccopigya in 
analysis (vi). In analysis (v), Euthyneura plus Cornirostra was a sister-group to all other 
gastropods except Coccopigya. In analysis (vii), five unexpected taxa (Nautilus, Nerita, 
Pterotrochus, Depressigyra and Coccopigya) disrupt the *apogastropod' lineage. None of 
the unexpected inclusions or exclusions had bootstrap support greater than 5096. 


Discussion 


The present analyses used data from six gene segments from four loci to address four ofthe 
major differences between morphological (Haszprunar 1988a; Ponder and Lindberg 1997) 
and molecular (Tillier et al. 1992, 1994; Rosenberg et al. 1997; McArthur and Koop 1999; 
Colgan et al. 2000; Harasewych and McArthur 2000) understanding of gastropod 
relationships. These were: (1) the position of Patellogastropoda; (2) the relationships of 
members of Cocculiniformia; (3) the relationships of members of Neritopsina; and (4) the 
monophyly of Heterobranchia. 

From the molecular perspective, the division of Gastropoda into Eogastropoda and 
Orthogastropoda remains an open question, despite the addition of more data in the present 
paper. The position of Patellogastropoda varies in present analyses as in previous molecular 
investigations (Rosenberg et al. 1997; McArthur and Koop 1999; Colgan et al. 2000). 
Long-branch length attraction (Felsenstein 1978; Lyons-Weiler and Hoelzer 1997; Siddall 
and Whiting 1999; Stiller and Hall 1999; Philippe and Germot 2000) may be a possible 
explanation for the pairing of Patellogastropoda with groups that would be unexpected on 
morphological grounds. Morphological support for the eogastropod/orthogastropod 
division is strong, but not incontestable. Character states supporting the division of 
Gastropoda into Eogastropoda and Orthogastropoda should be synapomorphic in the latter 
group and plesiomorphic or having an autapomorphy not derived from the state in 
Orthogastropoda in Patellogastropoda. The potential number of such characteristics is 
impressive. Fifteen changed state between the nodes uniting all gastropods and all 
orthogastropods in the Ponder and Lindberg (1997) topology. However, five have 
consistency indices less than 0.4 and one (adult operculum) is not applicable to 
Patellogastropoda, although they possess a larval operculum. Among the other nine 
characteristics, only three (18, the presence of a hypobranchial gland; 60, the bending plane 
of the radula; and 98, statocyst position) have possibly derived states in all major 
orthogastropod clades. In the remaining six characteristics, some or most Vetigastropoda 
share the same derived state as Patellogastropoda or have the symplesiomorphic state for 
Gastropoda. 

The situation with some characteristics is ambiguous; for example, with the 
hypobranchial gland. Sasaki (1998; his character 25) confirmed that a hypobranchial gland 
is absent in patellogastropods and noted its absence in Nautilus (although the nidamental 
glands may be homologous; Salvini-Plawen 1990). Sasaki (1998) also stated that the gland 
is absent in Fissurellidae on the basis of his own observations and Neomphalidae (fide 
McLean 1981), although it was recorded as present in these taxa by Ponder and Lindberg 
(1997). Fretter and Graham (1962) recorded a hypobranchial gland in Diodora and implied 
its presence in Emarginula (both fissurellids). Haszprunar (19895) observed a small 
hypobranchial gland in males of Pseudorimula (a fissurellid) but not in females. Israelsson 


140 Molluscan Research D. J. Colgan et al. 


(1998) recorded a hypobranchial gland in Pachydermia (related to Neomphalus) and one 
has also been recorded in Melanodrymia (Haszprunar 19894), but the presence or absence 
of one is not noted in Neomphalus by Fretter et al. (1981). Sasaki (1998) bases his statement 
regarding the absence of a hypobranchial gland on McLean (1981), who says that a thick 
folded gland, as seen in haliotids and trochids, is absent but that there are *...scattered 
subepithelial gland cells ... comparable to .... the Fissurellidae in which gland cells are 
present in the mantle skirt but do not form a discrete organ with a folded surface”. Given 
the presence of an undisputed gland in closely related taxa, what is present in Neomphalus 
is certainly a reduced hypobranchial gland, similar reductions being seen in many other 
gastropods, even within genera. These observations do not discount the possibility that the 
hypobranchial glands are secondarily absent in patellogastropods. They are absent in 
Scaphopoda, and possibly Cephalopoda, and a possibly homologous gland is present in 
Monoplacophora (Lemche and Wingstrand 1959; Wingstrand 1985; Haszprunar 1997). 

One of the characteristics supporting the monophyly of the orthogastropods, the 
flexoglossage condition of the radula (Haszprunar 1988a; Salvini-Plawen 1988; Ponder and 
Lindberg 1997), is now thought to be plesiomorphic, owing to some lateral bending of the 
radula being found in chitons (Guralnick and Smith 1999). Guralnick and Smith (1999) 
suggest that the stereoglossate condition of the radula in patellogastropods is secondary. 
The radular stroke of living monoplacophorans has not been examined, so the condition of 
their radula can only be inferred. However, Guralnick and Smith (1999) argue that it is also 
probably flexoglossate with the structure of the radula most like that of lepetid 
patellogastropods. Available data also suggest a flexoglossate condition in cephalopods, 
scaphopods and in ‘aplacophorans’ (Guralnick and Smith 1999). Thus, on the basis of these 
findings, the stereoglossate condition appears to be an autapomorphy of the 
patellogastropods. There are, however, some plesiomorphic states retained in the 
patellogastropod radula that are not found in other gastropods (Guralnick and Smith 1999). 

Since the publication of Ponder and Lindberg (1997), two new datasets add additional 
weight to the basal position of the patellogastropods. These relate to the buccal cartilages 
and the fine structure of the cephalic tentacles. 

Only the number of buccal cartilages present in the odontophore was scored by Ponder 
and Lindberg (1997). Sasaki (1998) and Guralnick and Smith (1999) have attempted to 
homologise the cartilages. Guralnick and Smith (1999) used position and shape as a 
primary means of tracking the evolution of the buccal cartilages. They argue that a medial 
pair of cartilages is plesiomorphic for Mollusca, as also (probably, therefore being 
secondarily absent in “aplacophorans”) are the dorsolateral (= anterolateral of Sasaki 
(1998)) cartilages. More likely, assuming 'aplacophorans' are basal (e.g. Haszprunar 
2000), the cartilages are probably synapomorphic of Testaria (sensu Haszprunar 2000). 
Whereas Sasaki (1998) considered these latter cartilages to be autapomorphies of 
patellogastropods, Guralnick and Smith (1999) argued that they were homologues of the 
dorsolateral cartilages of chitons and monoplacophorans. In these latter taxa, the two pairs 
of cartilages are attached by a connective tissue sheath, the space between being the hollow 
vesicles seen in those groups. Dorsolateral cartilages are absent in all Apogastropoda. A 
pair of dorsal cartilages is found in chitons and these are absent in modern 
Monoplacophora, but present in some patellogastropods. In addition, there are two pairs of 
posterior cartilages in chitons and the patellid patellogastropods (absent, presumably lost, 
in some of the more modified patellogastropods and in living Monoplacophora; Guralnick 
and Smith 1999). A single posterior pair is found in some vetigastropods and neritopsines. 
In patellogastropods, the subradular membrane is not associated with the medial cartilages 


Gastropod molecular phylogenetics Molluscan Research 141 


as it is in other gastropods, but is, instead, associated with the plesiomorphic dorsolateral 
(and dorsal cartilages when present), lying well above the medial cartilages. 

Künz and Haszprunar (2001) showed that the fine structure of the cephalic tentacles of 
patellogastropods differs significantly from that of vetigastropods and neritopsines and that 
they share ciliary features observed in bivalves and 'aplacophorans'. A similar 
configuration (stiff cilia with a more or less homogeneous pattern of microtubules) is 
unknown in most other gastropods, although somewhat similar cilia are known from the 
tentacles of the pulmonate Lymnaea (Emery 1992). In addition, the ciliary tufts of 
patellogastropods have several ciliary types, whereas in the other two groups the ciliary 
morphology is much more uniform. Further, patellogastropods, vetigastropods and 
neritopsines all show differences in their sensory elements, supporting and mucous cells. 

Other recent datasets that are less well resolved, but also appear to show that the 
patellogastropods are distinct from the vetigastropods and other gastropods, include larval 
musculature and the development of adult muscles (Wanninger et al. 1999) and sperm 
ultrastructure (e.g. Hodgson and Morton 1998). In other characteristics (e.g. cleavage 
pattern (van den Bigelaar and Haszprunar 1996), larval morphology and ciliation (Hadfield 
et al. 1997)), the patellogastropods and vetigastropods share assumed plesiomorphic 
conditions. 

A significant problem for the patellogastropod ancestors being the sister-group to the 
orthogastropods is the lack of undoubted patellogastropods or obvious coiled ancestors in 
the early fossil record. The oldest undoubted patellogastropod has been confirmed recently 
(on the basis of shell structure) from the Triassic (Hedegaard ef al. 1997). Recognition of 
such, probably coiled, ancestors will be difficult, but Wagner (2002) very tentatively 
suggests ‘euomphalinaes’ as candidates. If this was the case, the split in the two main 
gastropod lineages occurred in the Late Cambrian, around 510 million years ago. 

Cocculiniformia are not monophyletic in our analyses. Notocrater (Pseudocculinidae) is 
strongly associated with Vetigastropoda, as found by Ponder and Lindberg (1997). 
Coccopigya is variously associated in derived positions (e.g. with Heterobranchia or 
Depressigyra and Perotrochus). The pairing with Nerita at the base of Orthogastropoda 
observed by Ponder and Lindberg (1997) is not found in any of our analyses. There are no 
morphological characteristics directly suggesting that Cocculinidae and Heterobranchia are 
sister-groups, although the sinistral protoconch coiling found in all members of the latter 
group has its analogue in at least some Cocculinidae. The pairing of Coccopigya with two 
other deep-sea taxa (Depressigyra and Perotrochus) appears to be coincidental because 
Notocrater and Lepetodrilus are also found in this environment. 

Heterobranchia is rarely monophyletic in our analyses, owing to the variable placement 
of Philippea. Euthyneura is monophyletic in all analyses. Within Euthyneura, 
Opisthobranchia and Pulmonata are rarely monophyletic, concurring with Dayrat ef al. 
(2001). They found that Opisthobranchia is paraphyletic with respect to Pulmonata, albeit 
that the nodes suggesting this observation had low bootstrap support. 

The clade comprising Euthyneura plus Cornirostra is strongly supported in our analyses, 
supporting the suggestion of Haszprunar (19884) that Valvatoidea is closer to Euthyneura 
than is Architectonicoidea. Confirmation of this will require data from other members of 
the family because Philippea is undoubtedly highly autapomorphic and its placement 
appears to depend on long-branch attraction. Questions of monophyly or paraphyly of 
Heterostropha, which includes the well-established families | Omalogyridae, 
Pyramidellidae, Valvatoidea, Architectonicoidea and Rissoellidae, as well as a number of 
recently created Recent and fossil families, will be a fruitful area for further research. 


142 Molluscan Research D. J. Colgan et al. 


The relatively large genetic differentiation of Heterobranchia in maximum parsimony 
trees suggests that the clade has a long evolutionary history, particularly if substitution rates 
are even remotely clocklike. The genetic distinction of Heterobranchia is emphasised by 
mitochondrial DNA genome organisation. In studied Euthyneura (for references, see 
Kurabayashi and Ueshima 2000a), this is radically different to the arrangement in the 
caenogastropod Littorina (Widling et al. 1999) but similar to that of Omalogyra 
(Kurabayashi and Ueshima 20005). Other gene order work on opisthobranchs (Grande 
2001; Medina et al. 2001) has yet to be reported in full. To date, the gene order data are 
based on an extremely small sampling and whether or not Littorina is typical of 
caenogastropods is unknown. For example, Collins et al. (2001) and Rawlings et al. (2001) 
report a major gene order rearrangement within the caenogastropod Vermetidae. Sperm 
structure (Healy 1993) also supports the monophyly of heterobranchs as a whole and 
Euthyneura. The earliest undoubted heterobranch fossils date from the early Devonian 
(390-408 million years ago; Fryda and Blodgett 2001), although some taxa included in the 
subulitoideans are likely heterobranchs and this grouping extends into the Ordovician 
(Nützel et al. 2000). 

The affinities of the Heterobranchia (excepting Philippea) are with the Caenogastropoda 
in a recognisably ‘apogastropodan’ group (Salvini-Plawen and Haszprunar 1987; 
Haszprunar 1988a; as extended by Ponder and Lindberg 1997), although some taxa are 
anomalously included or excluded in some analyses. This contrasts with Colgan et al. 
(2000), where the ‘apogastropod’ group also had unexpected inclusions (Nerita and 
Nautilus) and Caenogastropoda and Heterobranchia were intermixed. Apogastropoda are 
monophyletic and comprised of monophyletic Caenogastropoda and Heterobranchia in 
McArthur and Koop (1999) and Harasewych and McArthur (2000), although these studies 
include fewer taxa from these latter groups. 

Caenogastropoda is monophyletic with the exception of the anomalous inclusion of 
Nerita and/or Nautilus in some analyses and the exclusion of Leptopoma and Cypraea in 
analyses (i) and (vii). Relationships within Caenogastropoda are not well resolved in the 
present analyses. In particular, although various sets of two of the five genera of 
Neogastropoda included in the dataset are found in monophyletic clades in some analyses 
and four of the five are grouped in the Bayesian analysis (xi), this morphologically strongly 
supported group is not otherwise shown as closely related, as also found by Harasewych 
et al. (1997b). 

Architaenioglossa comprise a number of superfamilies (previously two, now three) not 
considered close relatives by Ponder and Lindberg (1997). The taxa included here, 
Bellamya representing Vivipariodea (previously included within what is now considered to 
be a separate superfamily Ampullarioidea) and Leptopoma representing Cyclophoroidea, 
are monophyletic only in analysis (ix) based on maximum likelihood and analysis (xi) based 
on Bayesian likelihood. Bellamya is a member of the Caenogastropoda in all our analyses, 
but the position of Leptopoma varies widely although remaining within an apogastropodan 
group, except when all data are considered (analysis (i)), where it is a sister-group to an 
heterogeneous taxon (Fig. 2). Campanile is always associated with Bellamya or Leptopoma. 
McArthur and Koop (1999), using partial 28$ rDNA sequences, also found that the 
architaenioglossans (Ampullaria and Viviparus) were not monophyletic and, unlike our 
result, that Ampullaria and Campanile were sister-taxa. In their analyses, and with most of 
our analyses, the architaenioglossans lie within the caenogastropods, as suggested by 
Ponder and Warén (1988) and demonstrated in the morphological analyses of Ponder and 
Lindberg (1996, 1997). Alternative hypotheses have been produced on the basis of 


Gastropod molecular phylogenetics Molluscan Research 143 


morphological analyses, notably suggesting that the architaenioglossans are the 
sister-group to the apogastropods and part of a paraphyletic ‘Archaeogastropoda’ 
(Haszprunar 1988a) or that they belong to a clade (together with Neritopsina and 
Neomphaloidea), which is sister-group to the caenogastropods (Barker 2001). 

The placement of the Neritopsina (or Neritimorpha) remains uncertain. This group, plus 
the Cocculiniformia (among our studied taxa), formed a sister-clade to all other 
Orthogastropoda in Ponder and Lindberg’s (1997) preferred topology and in Rosenberg 
et al. (1997), although in this latter analysis the patellogastropod was included within the 
apogastropods. In the morphological analysis undertaken by Sasaki (1998), the 
patellogastropods formed the base of the gastropod clade and Cocculina appeared within a 
clade containing Neomphalus and the vetigastropods. Neritopsines formed one of four 
branches in an unresolved Gastropoda in the analysis of Harasewych et al. (1997a) and one 
branch of a basal trichotomy in McArthur and Koop (1999: fig. 3) and our analysis (vi). As 
in analyses (ii), (iv) and (viii), the Neritopsina was a sister-group to all other gastropods in 
maximum parsimony analyses (excluding one 25 bp insert) of the 18S rDNA data of 
Harasewych and McArthur (2000: fig. 24) and their maximum likelihood analysis (fig. 
2C). When two longer inserts were excluded, Neritopsina was a sister-group to 
Vetigastropoda, this pair being a sister-group to Apogastropoda (Harasewych and 
McArthur 2000: fig. 23). Nerita was placed within Caenogastropoda in Colgan et al. 
(2000), in accordance with our analyses (i), (v), (vii), (ix) and (xi) but in contrast with all 
recent morphological assessments (cf. Bieler 1992). In Barker's (2001) morphological 
analysis, Neritopsina was the sister-group to a clade consisting of Neomphaloidea + 
Architaenioglossa. This combination was a sister-group taxon to the caenogastropods. In 
our analyses (iii) and (vi), Nerita is a sister-group to Apogastropoda, allowing the 
possibility that larval planktotrophy arose once only in gastropods (cf. Ponder 1991; see 
also discussion in Ponder and Lindberg 1997: 209—213; and Fryda 2001). 

The strict consensus of the maximum parsimony trees from analysis (vi) has notable 
similarities to recent morphological hypotheses. Agreeing with Ponder and Lindberg 
(1997), Caenogastropoda and Heterobranchia are monophyletic, as is Vetigastropoda, with 
the predicted inclusion of Notocrater. Patellogastropoda is basal, although excluded from 
Gastropoda. 

Although the results ofall analyses should be included in discussions of the phylogenetic 
implications of our data, we give a little more weight, when results differ, to those of 
analysis (vi), where, with Nautilus as the only outgroup, third-position data are excluded 
and transitions and transversions are weighted differently (Fig. 5). Comparison of 
consistency indices supports the exclusion of data because they are higher in trees including 
third positions than those excluding them. As judged by the consistency indices, excluding 
these data reduces the amount of phylogenetic noise. Arguing for differential weighting is 
the low transition to transversion ratio in the overall data for coding genes CO7 and H3. 
This ratio increases when third positions are excluded, indicating a substantial degree of 
saturation. This analysis (as well as some others) has a high probability of homogeneity of 
phylogenetic inferences from the separate gene data. 

When the chiton and bivalves are included, the outgroups are not monophyletic in any 
analysis. The use of Nautilus as the sole outgroup is suggested by the consensus on 
morphological grounds that Cephalopoda or Monoplacophora are the sister-taxon to 
Gastropoda (reviewed by Ponder and Lindberg 1997). Although not included in our 
analysis, scaphopods have recently been shown to be the sister-taxon to the cephalopods 
(Waller 1998; Haszprunar 2000; Giribet and Wheeler 2002; Wanninger and Haszprunar 


144 Molluscan Research D. J. Colgan et al. 


2002), in contrast with earlier hypotheses that linked them to the bivalves. This relationship 
is, however, not apparent in the analysis of Rosenberg et al. (1997). 

Unfortunately, despite considerable advancement in our knowledge of Palaeozoic fossils 
in the past decade, the origins of the major gastropod groups remain obscure, although all 
should have differentiated by the early Ordovician shortly after gastropods evolved (Wagner 
2001, 2002). Whereas the considerable extinctions that have occurred during gastropod 
evolution may account for some ofthe long branch attraction issues encountered (especially 
between the patellogastropods and the remainder of the gastropods), breaking down some 
of the long branches encountered in this dataset by the addition of more taxa (Graybeal 
1998) may be possible. 

Despite more molecular data having been incorporated in these present analyses, some 
major aspects of gastropod phylogeny remain equivocal. Additional genes, gene order data 
and more refined morphological data will be required to resolve many of these issues, as 
well as better data on Palaeozoic gastropods. 


Acknowledgments 


We are grateful to Diana Picone for collecting more than half the U2 sequences and to the 
following for providing samples: M. G. Harasewych for tissues of Notocrater, Nautilus and 
Perotrochus; A. G. McArthur for providing DNA of the vent taxa Lepetodrilus and 
Depressigyra (with the kind permission of Dr V. Tunnicliffe); B. Marshall for Coccopigya; 
Fred Wells for Campanile; and Rosalyn Yardin for Anadara DNA. P. Eggler, I. Loch, P. 
Colman, A. Miller and S. Clark assisted in the collection of material. 


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http://www.publish.csiro.au/journals/mr 


CSIRO PUBLISHING 
www.publish.csiro.au/journals/mr Molluscan Research, 2003, 23, 149-158 


Reassessment of Australia's oldest freshwater snail, Viviparus (?) 
albascopularis Etheridge, 1902 (Mollusca: Gastropoda: Viviparidae), 
from the Lower Cretaceous (Aptian, Wallumbilla Formation) of White 
Cliffs, New South Wales 


Benjamin P. Kear^, Robert J. Hamilton-Bruce^*, Brian J. Smith" and Karen L. 
Govvlett-Holmes? 


ASouth Australian Museum, North Terrace, Adelaide, SA 5000, Australia. 

BSchool of Biological, Earth and Environmental Science, University of New South Wales, Sydney, NSW 
2052, Australia. 

CQueen Victoria Museum, Wellington Street, Launceston, Tasmania 7250, Australia. 

DCSIRO Division of Marine Research, GPO Box 1538, Hobart, Tasmania 7001, Australia. 

FTo whom correspondence should be addressed. Email: hamilton-bruce.robert@saugov.sa.gov.au 


Abstract 


Viviparus (?) albascopularis Etheridge, 1902 is Australia’s oldest documented fossil freshwater gastropod. 
The taxon was established on the basis of a single opalised shell from the Lower Cretaceous (Aptian) marine 
deposits of the Wallumbilla Formation (Doncaster Member) at White Cliffs, New South Wales. 
Reassessment indicates that original placement in the caenogastropod family Viviparidae is justified; 
however, the specimen is reassigned to the endemic Australian genus Noropala Cotton, 1935 on the basis 
of shell morphology and close morphometric similarity to extant species. Implications for the origins and 
current distribution of Australian viviparid taxa are discussed. 


Introduction 


The fossil record of Australian Cretaceous non-marine gastropods is depauperate and very 
poorly known. Nearly all the currently identified material has been recovered from the 
middle-upper Albian (Lower Cretaceous) fluviatile-lacustrine deposits of the Griman Ck 
Formation at Lightning Ridge in north-western New South Wales (NSW). This assemblage 
comprises primarily viviparids, although numerous other groups, including thiarids 
(Hamilton-Bruce ef al. in press), succinids, camaenids (currently under study) and 
amphibolids (B. J. Smith, R. J. Hamilton-Bruce and B. P. Kear, unpublished data) are also 
present (Dettman et al. 1992; Smith 1999; Hamilton-Bruce ef al. 2002). The only other 
documented Australian Cretaceous non-marine gastropod fossil is a single opalised shell 
(AM F17456) from the Aptian (Lower Cretaceous) marine sediments of the Wallumbilla 
Formation (Doncaster Member) at White Cliffs, NSW (Burton and Mason 1998: see figs 
1,2 for detailed geological and locality maps of the area). This specimen was described 
(from a private collection belonging to a Mr H. Y. L. Brown of Adelaide, later acquired by 
the Australian Museum) by Etheridge (1902) and tentatively assigned to Viviparus (?) 
albascopularis, recognising similarity to members of the currently extant caenogastropod 
family Viviparidae. The present paper provides a revised description of the holotype (AM 
F17456) and only known specimen of K (?) albascopularis Etheridge and reinterprets its 
taxonomic placement. Implications for the origins and distribution of Australian viviparid 
taxa are discussed. 

The Lower Cretaceous (Aptian) opal-bearing deposits of White Cliffs have long been 
known as a productive locality for fossils. Anderson (1892) briefly remarked on the 
presence of mollusc remains, crinoids and wood. Jaquet (1893) recorded belemnitid 


© Malacological Society of Australasia 2003 10.1071/MR03003 1323-5818/03/020149 


150 Molluscan Research B. P. Kear et dl. 


cephalopods and Devonian invertebrate fossils preserved as impressions in large erratic 
clasts. These were interpreted as a product of reworking from underlying Palaeozoic 
conglomerates. Etheridge (1897, 1902, 1904) reported the occurrence of bivalves, 
ammonites, naticid gastropods, plesiosaurs and ichthyosaurs. More recent studies by White 
(1926), Molnar (1980, 1991) and Kemp (1991) have also identified lungfish and dinosaur 
remains. 

Viviparus (?) albascopularis Etheridge is currently the oldest known Australian fossil 
freshwater gastropod and one of the earliest members of the Viviparidae. Today, this 
cosmopolitan family comprises various taxa, characterised by medium-large-sized 
turbiniform shells, possessing a rounded body whorl, moderately high and pointed spire, 
wide, round aperture and a concentric, horny operculum (Smith 1992). Within Australia, 
the distribution of the group is limited to a few species occurring in the large drainage 
basins that span much of the arid centre, northern tropical and coastal regions. 

The fossil record for Viviparidae is known from the Jurassic-Recent (Viviparus Montfort, 
1810), with a tentative report based on an internal shell mold (?Bernicia Cox, 1927), possibly 
of marine origin, from the Early Carboniferous of England (Brookes-Knight ef al. 1960). 
The group's Australian record is very sparsely documented. Cotton (1935) described a 
species of Notopala Cotton, 1935 (N. wanjacalda) from upper Pleistocene sediments along 
the Murray River, near Sunnyside, South Australia (SA), and noted a second taxon 
(Notopala sp.) from the same deposit, which showed strong similarity to the extant N. hanleyi 
(von Frauenfeld, 1862). Dettman et al. (1992) reported viviparid snail shells from the Lower 
Cretaceous (middle-upper Albian) deposits of Lightning Ridge, NSW, as did Smith (1999), 
who also recorded representatives of the Naticidae, Thiaridae and Ellobiidae. Recently, 
Hamilton-Bruce et al. (2002) described a new genus (Albianopalin) and two new species 
of viviparid from Lightning Ridge, as well as indeterminate material attributable to the 
currently extant endemic Australian taxon Noropala. Other Australian non-marine 
gastropod fossils (all of Tertiary age) have been documented by Chapman (1937), 
McMichael (1968), Archer et al. (1994), Arena (1997) and Pledge et al. (2002). Occurrences 
from elsewhere in Australasia are rare, particularly in Cretaceous sediments. Some of the 
few examples include viviparids (genera uncertain) and thiarids (?Melanoides Olivier, 1804) 
from the Cenomanian-?Santonian (Upper Cretaceous) of New Zealand (Henderson ez al. 
2000) and possible thiarids (Pyrgulifera Meek, 1871) from both the Campanian-lower 
Maastrichtian (Upper Cretaceous) of the Chatham Islands (Stilwell 1998) and ?Campanian 
of New Caledonia (Henderson ef al. 2000). 


Material and methods 


Material registered as the holotype of V (?) albascopularis Etheridge includes a single shell with broken 
aperture margin and protoconch (AM F17456), preserved entirely in potch (non-precious or common opal). 
The specimen is derived from an unknown mine locality in the opal-bearing deposits of White Cliffs near 
Wilcannia in north-western NSW. The lithostratigraphic nomenclature for Lower Cretaceous rocks of the 
White Cliffs area was recently discussed by Burton and Mason (1998), who placed them within the 
Doncaster Member of the Wallumbilla Formation (Eromanga Basin), a unit of Aptian-middle Albian 
(115-approximately 100 million years ago; sensu Lowrie et al. 1980) age. However, the White Cliffs 
opal-bearing sediments are regarded as representing only the lower Aptian section of the Doncaster 
Member and comprise predominantly sandy/silty claystone and fine-grained sandstones deposited in a 
near-shore coastal marine setting (Burger 1988; Burton and Mason 1998). Determinations of palaeolatitude 
place the White Cliffs area as high as 70°S during the Early Cretaceous (Embleton 1984). Palaeoclimatic 
indicators for the region also suggest predominantly cool, strongly seasonal conditions with winter freezing 
(Frakes and Francis 1988, 1990; Sheard 1990; Frakes et al. 1995; De Lurio and Frakes 1999; Henderson 
etal. 2000). Estimates of sea level isotopic palaeotemperatures in the south-western section of the 


Reassessment of Viviparus (?) albascopularis Etheridge Molluscan Research 151 


Eromanga Basin have yielded averages as low as 12.2°C (Stevens and Clayton 1971; Dettman ef al. 1992). 
However, Selwood et al. (1994) reported revised isotopic data supporting much cooler ocean temperatures 
during the Early Cretaceous. Indeed, Pirrie et al. (1995) indicated palaeotemperatures of around 109C based 
on Early Albian belemnites from the Carnarvon Basin, Western Australia (situated at approximately 45° 
palaeolatitude during the Cretaceous). In contrast, Huber et al. (1995) and Huber and Hoddell (1996) 
argued that minimal pole-to-equator thermal gradients existed during much of the Middle-Late Cretaceous. 
This was also discussed by Henderson et al. (2000), who noted that although palaeotemperatures at 707-809 
latitude would certainly have been more equitable than they are today, evidence such as the distinct 
growth-banding in Australian Cretaceous wood (Dettman et al. 1992), and the presence of potentially 
ice-rafted quartzite/porphyritic boulders (Frakes and Francis 1988, 1990; Frakes er al. 1995) and 
glendonites (crystal aggregates pseudomorphing the calcium carbonate hexahydrate mineral ikaite; Sheard 
1990; DeLurio and Frakes 1999) attests to the strong seasonality and winter freezing along the inboard 
extremity of the Australian epicontinental seaway during the Aptian. 

Shell diameters were measured using the method of Boycott (1928), defined as ‘... the greatest 
dimension that can be found starting with the edge of the lip to a point on the opposite side of the shell on 
the last whorl’. Shell measurements were made to the nearest 0.05 mm using dial calipers. 

The Australian Museum, Sydney is abbreviated as AM throughout. 


Systematics 
Class GASTROPODA 
Superorder CAENOGASTROPODA 
Superfamily VIVIPARIOIDEA 
Family VIVIPARIDAE Gray, 1847 
Diagnosis 


Medium to large dextral, turbiniform shells with rounded body whorl, spire moderately 
high and pointed, aperture wide and round to distinctly lunate—ovate, lirae present or absent 
and horny, concentric operculum. 


Remarks 


The above diagnosis follows Smith (1992), modified to accommodate the presence of a 
distinctly lunate-ovate aperture and spiral lirae on the last body whorl of the holotype 
specimen, AM F17456. Viviparid snails are, as their name suggests, live bearing and are 
found in both lotic and lentic systems throughout the world (Browne 1978). Within 
Australia, the family is currently represented by the extant native genera Notopala, Larina 
Adams, 1851 and Centrapala Cotton, 1935 (see Smith 1992) and the introduced Bellamya 
heudei guangdungensis (Kobelt, 1906), an Asian species now recorded in the wild in NSW 
(Shea 1994). 

Australian endemic viviparids have undergone extensive taxonomic revision in recent 
years (the most inclusive analysis currently being undertaken by W. Ponder of the 
Australian Museum, personal communication, 2002), with better understanding of 
intraspecific shell variation and morphometric data resulting in a substantial reduction in 
the number of accepted species (see Sheldon and Walker 1993). However, many of the key 
characteristics used to determine interrelationships, such as shell colour and form of the 
operculum, are usually lost in fossil material; therefore, only structural features of the shell 
(see below) can be used to assign them to taxa. 


152 Molluscan Research B. P. Kear et al. 


Genus Notopala Cotton, 1935 


Notopala Cotton, 1935: 339. Type species (by original designation): Paludina hanleyi von Frauenfeld, 
1864. 


Diagnosis 


Shell dextral, globose-conic, subumbilicate, up to 5 whorls, ventricose to angulate below 
the periphery; spiral lirae present on periostracum of last body whorl in some taxa; aperture 
large and subovate to lunate-ovate, aperture size equal to or greater than height of spire, 
operculum corneous (unknown in fossil taxa). 


Remarks 


Despite the recovery of AM F17456 from a deposit of marine origin, its shell morphology 
fits within the currently accepted diagnosis of Notopala and Viviparidae with only slight 
modification (the presence of a distinctly lunate—ovate aperture and calcified spiral lirae on 
the last body whorl). To justify placement within the family and to establish a basis for both 
generic reassignment and comparison with extant species, we have applied parts of the 
morphometric data gathered by Sheldon and Walker (1993). The histogram in Fig. 1 is 
based on measurements of shell characteristics for each of the living Australian species of 
Notopala (derived from Sheldon and Walker 1993) and illustrates the morphometric 
similarity of AM F17456, redescribed herein, to currently existing members of the genus. 


Notopala albascopularis (Etheridge, 1902) 
(Fig. 24-D) 


Viviparus (?) albascopularis Etheridge, 1902: 43; pl. VII, figs 8,9. 


Material examined 


Holotype. AM F17456. Type locality and horizon: White Cliffs Opal Field (exact mining claim 
locality unknown), near Wilcannia, north-western NSW. The deposits form part of the Doncaster Member 
ofthe Wallumbilla Formation (Rolling Downs Group), Eromanga Basin, and are of Aptian age (see Burton 
and Mason 1998). This corresponds to the Cyclosporites hughesii-lower-most Crybelosporites striatus 
spore-pollen Zones and Odontochitina operculata-Diconodinium davidii dinoflagellate zones of Helby 
et al. (1987). 


Diagnosis 


With the features of the genus; irregular, pustular lirae present on the last body whorl; 
aperture somewhat poorly preserved but distinctly lunate-ovate and markedly adapically 
situated. 


Description 


Shell (Fig. 24—C) dextral, turbiniform, very slightly carinate, subglobose, 21.65 mm high, 
20.05 mm maximum diameter, 13.55 mm aperture length, 11.5 mm aperture width, 8.1 mm 
spire length. Teleconch four complete whorls and broken parts indicating further whorls, 
probably up to five. Whorls impressed. Relatively evenly spaced fine spiral prosocline 
growth lines on lower three whorls. Aperture large (13.55 mm high), slightly subangular, 
distinctly ovate-lunate, markedly adapically situated. Opal replacement of original shell 
material indicates 12 or more irregular, pustular lirae on the last body whorl (Fig. 2D). 


Reassessment of Viviparus (?) albascopularis Etheridge Molluscan Research 153 


APL SHW APW 


4.0 
3.0 
2.0 
1.0 
0.0 Eli İd 
i? "Banded" Shells N. waterhousii 
ER Notopala sublineata (Cooper) HE N. essingtonensis 
ll Notopala sublineata (Darling) EH N.alisoni 7 
SN. hanleyi N. albascopularis 
| = Standard deviation * single sample 


Fig. 1. Histogram showing results of morphometric analysis. Taxa include living species of Notopala 
(measurements modified from Sheldon and Walker 1993) and N. albascopularis (Etheridge, 1902), AM 
17456. APL, aperture length; APW, aperture width; SHW, shell width. 


Remarks 


Holotype unique. The presence of irregular, pustular lirae on the last body whorl and a 
distinctly lunate-ovate, markedly adapically situated aperture separates N. albascopularis 
(Etheridge, 1902) from other species of Notopala. The generic reassignment of the 
holotype specimen is highly significant because previously none of the currently living 
native Australian viviparid genera was known from deposits older than middle-upper 
Albian (upper-most Lower Cretaceous). This temporal range is now extended back to the 
Aptian. 


Discussion 


The fossil record of Australian non-marine gastropods is very sparsely documented, with 
the majority of existing reports describing material of Tertiary to Holocene age. Mesozoic 
specimens have, to date, only been identified from middle to upper Albian (Lower 
Cretaceous) sediments of the Griman Ck Formation at Lightning Ridge in northern NSW 
(Dettman et al. 1992; Smith 1999; Hamilton-Bruce ef al. 2002). Therefore, recovery of 
N. albascopularis (Etheridge, 1902) from the Aptian deposits of the Wallumbilla 
Formation (Doncaster Member) at White Cliffs gives this taxon the distinction of being 
both Australia's oldest definitively assigned non-marine gastropod and the earliest recorded 
representative of the Viviparidae in Australia. Recognition of N. albascopularis as a 
viviparid also serves to extend the family's temporal range in this region back to at least the 
later stages of the Early Cretaceous and, given its Jurassic-Recent fossil record in Europe, 
suggests a possible pre-Jurassic Pangean origin for the group. By the Early Cretaceous, the 
family had clearly diversified within the Gondwanan region, establishing key endemic taxa 
that still exist as descendant lineages today. This may be seen, to some extent, in the strong 
morphological similarity between N. albascopularis and other currently extant Australian 


154 Molluscan Research B. P. Kear et al. 


10 mm 


Fig.2. AM 17456 Notopala albascopularis (Etheridge, 1902) in (A) apertural, (B) dorsal and (C) apical 
views. (D) Magnified section of last body whorl showing lirae (arrowed). 


Reassessment of Viviparus (?) albascopularis Etheridge Molluscan Research 155 


species of Notopala, particularly N. hanleyi and N. sublineata (Conrad, 1850) (see Fig. 1), 
both of which could potentially represent morphological derivatives. However, further 
study and the discovery of additional fossil material are required before any definitive 
phylogenetic relationship can be demonstrated. 

Although there are numerous records of Cretaceous freshwater bivalves from Australia 
(McMichael 1957; Ludbrook 1985; Jell and Duncan 1986; Dettman et al. 1992; Hocknull 
1997), there are very few for non-marine gastropods of the same period. Indeed, most of the 
better documented terrestrial units, including the Wonthaggi Formation, Eumeralla 
Formation and Koonwarra Beds (Korumburra Group) of Victoria (see Jell and Duncan 1986; 
Rich et al. 1988; Rich and Rich 1989) and Winton Formation of Queensland and SA (see 
Ludbrook 1985; Dettman et al. 1992; Hocknull 1997), have yet to produce any identifiable 
gastropod remains. The reasons for this apparent absence are unknown, but could be related 
to preservational biases, such as shells rapidly breaking up or dissolving after death. 
However, isolated specimens, such as the holotype of N. albascopularis, seem to have, on 
occasion, survived transport over considerable distances (perhaps on floating vegetation) 
prior to eventual burial. This scenario also appears to have been common for many of the 
other fossils at White Cliffs, which include a high proportion of terrestrial plant remains 
(Anderson 1892; Jaquet 1893; Etheridge 1902; Newton 1914), freshwater invertebrates 
(Etheridge 1902; Newton 1914; McMichael 1957; Dettman ef al. 1992) and occasional 
freshwater/terrestrial vertebrates (White 1926; Molnar 1980, 1991; Kemp 1991), all 
probably derived from fluviatile input into the near-shore marine depositional environment. 

Another factor possibly influencing the distribution of Early Cretaceous freshwater 
gastropods in Australia may have been the strongly seasonal cool to cold climates, which 
characterised many of the high-latitude continental (Douglas and Williams 1982; Gregory 
et al. 1989; Dettman et al. 1992; Cantrill 1998) and marine (Frakes and Francis 1988, 1990; 
Sheard 1990; Frakes er al. 1995; De Lurio and Frakes 1999) environments of the time. 
Although this may have limited the number of available habitats for non-marine gastropod 
species, it does not appear to have restricted overall taxonomic diversity or specimen 
numbers in the few deposits where they occur. Indeed, it is interesting to note that in the 
Lower Cretaceous freshwater river and lake deposits of Lightning Ridge, viviparids are one 
of the most common invertebrate faunal elements, far outnumbering other sympatric 
groups, such as thiarids, ellobiids and naticids (Smith 1999). The reasons for this apparent 
success are unknown, but could be related to the ability ofthe viviparids to give birth to live 
young and, thus, secure a competitive advantage over their contemporaries (however, larval 
brooding is also present in thiarids). Similarly, strict adaptation to freshwater may have 
enabled Cretaceous viviparids to rapidly colonise available upstream habitats. This 
contrasts with naticids and thiarids, whose Cretaceous record is largely derived from 
near-shore marine strata (see Etheridge 1902, Ludbrook 1966; Dettman et al. 1992; Stilwell 
1998; Henderson ef al. 2000), and may reflect a preference for more brackish water 
conditions around estuaries and coastal lagoons. 


Acknowledgments 


We thank Robert Jones for generous provision of AM F17456 for study. Philip Ryan 
examined the statistical methods used and Chris Izzo assisted with measurements and 
statistical data analysis. This manuscript benefited greatly from the comments of Winston 
Ponder and Jeffrey Stilwell. The South Australian Museum, Origin Energy, The Advertiser, 
Coober Pedy Tourism Association and the Waterhouse Club contributed financially to this 
project. 


156 Molluscan Research B. P. Kear et al. 


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http://www.publish.csiro.au/journals/mr 


CSIRO PUBLISHING 
www.publish.csiro.au/journals/mr Molluscan Research, 2003, 23, 159—178 


Relationships of Placostylus from Lord Howe Island: an investigation 
using the mitochondrial cytochrome c oxidase 1 gene 


Winston E Ponder, Donald J. Colgan^, Dianne M. Gleeson? and Greg H. Sherley" 


AAustralian Museum, 6 College Street, Sydney, NSVV 2010, Australia. 

BEcological Genetics Laboratory, Landcare Research, PB 92170, Auckland, Nevv Zealand. 
CDepartment of Conservation, PO Box 12-416, Wellington, New Zealand. 

>To whom correspondence should be addressed. Email: winstonp@austmus.gov.au 


Abstract 


Large (5-9 cm in length) land snails of the genus Placostylus are found in New Caledonia and the Loyalty 
Islands, northern New Zealand, the Three Kings Islands just north of New Zealand and on Lord Howe 
Island. Their presence on Lord Howe, an oceanic island less than 6 million years old, has been an intriguing 
biogeographical question. Maximum parsimony and maximum likelihood analyses using cytochrome c 
oxidase subunit I sequence data suggest that the Lord Howe Island and mainland New Zealand taxa are 
sisters, but that the Three Kings taxon is independently derived, possibly from New Caledonian stock. 
Placostylus colonies throughout the area of the present study are under considerable threat, with many 
intraspecific forms and some species threatened and some listed as endangered species. The taxonomic and 
conservation status of the Lord Howe Island populations are discussed. 


Additional keywords: biogeography, New Caledonia, New Zealand, south-west Pacific, systematics. 


Introduction 


Oceanic islands typically have high levels of endemism and the origin of their species 
continues to be an intriguing question for evolutionary biologists. Notable examples among 
vertebrates of such studies include Darwin's finches of the Galapagos Islands, the 
honeyeaters of Hawaii (Freed et al. 1987) and Brachylophis on Pacific Islands (Gibbons 
1981, 1985). There are also spectacular examples of radiations among invertebrates, some 
of the better known being the Pacific Island land snails, where there have also been massive 
human-induced extinctions (e.g. Solem 1990; Cowie 1992, 1996). 

The likelihood of dispersal of individuals and taxa to oceanic islands is dependent on 
many factors, including the distance from source populations and the size and habitat 
complexity of the island. However, overriding all these factors is the dispersal ability of the 
taxon. Dispersal ability can be determined by intrinsic abilities (power of flight, body size 
and physiology (e.g. resistance to desiccation, tolerance of salt water, habits or habitat 
preferences; an arboreal species may be more likely to be transported by wind storms than 
a ground-living or burrowing species)) or extrinsic factors (prevailing winds, presence of 
suitable dispersal agents etc.). Land snails have no intrinsic means of long-distance 
dispersal, although small-sized species, in particular, may be dispersed aerially during 
major storms, accidentally carried by birds (Rees 1965; Vagvolgyi 1975; Kirchner et al. 
1997) or rafted on floating vegetation. Successful long distance passive dispersal for most 
taxa is rare, especially once communities are established (Ward and Thornton 2000). The 
improbability of oversea dispersal for some taxa has led to hypotheses involving sunken 
continents or land bridges. 


© Malacological Society of Australasia 2003 10.1071/MR03001 1323-5818/03/020159 


160 Molluscan Research W. Ponder et al. 


P. fibratus 
and five others 


P. bivaricosus 
Lord Howe Island 


P. bollonsi». 


P. ambagiosus 
P. hongii 


ə 
New Zealand 


Fig. 1. The SW Pacific, showing the locations of the species of Placostylus included in the present 
analysis. The ellipse around New Caledonia is intended to show the overall range of the six species of 
Placostylus recognised from that area. The diamond indicates the locality of the New Caledonian 
specimen of P. fibratus used in the analysis. 


Lord Howe Island (31?33'S, 159°05 E) lies in the Tasman Sea, is approximately 11 km 
long and rises to 875 m. It is nearest to eastern Australia, lying 700 km NE of Sydney and 
496 km E of Port Macquarie, the nearest point on the coast of New South Wales (NSW). 
Norfolk Island is 890 km away and Auckland, New Zealand, is 1560 km away (Fig. 1). The 
highly endemic fauna and flora have elicited much discussion (see Paramonov 1958, 1960, 
1963). The island is of volcanic origin, being formed between 6.4 and 6.9 million years ago 
(McDougall et al. 1981), and lies on the eastern edge of the Lord Howe Rise, a submarine 
fragment of eastern Australia that separated in the Cretaceous (Cook and Belbin 1978). 
Drill cores show that the Rise itself has never been above water (Van der Lingen 1973). A 
chain of submarine seamounts runs due north of Lord Howe Island. These are assumed to 
be progressively older northwards (McDougall ef al. 1981). 

Among the highly endemic fauna and flora is the extinct horned tortoise (Meiolania 
platyceps Owen, 1886) that was apparently terrestrial with poor swimming abilities 
(Gaffney 1983, 1996) and with close relationships to taxa in eastern Australia and New 
Caledonia (Walpole Island; Gaffney 1996). Notable among the endemic flora are three 
endemic genera of palms. There are many indigenous invertebrates, including over 80 
species of land snails (Iredale 1944). Prominent among the land snails is P/acostylus 
bivaricosus (Gaskoin, 1855), which reaches up to approximately 8 cm in length. Hedley 
(1892) stated that “... Placostylus appears a more fruitful subject of study [for 
biogeography] than any other molluscan genus inhabiting the same area' and the mystery 
of the presence of Placostylus on Lord Howe was also discussed by Etheridge (1891). 


Placostylus relationships based on mtDNA Molluscan Research 161 


The distribution of Placostylus was such a conundrum to Hedley (1892) that he 
hypothesised that the islands containing these snails were “... portions of a shattered 
continent [which he called the Melanesian Plateau] and are connected by shallow banks 
formerly dry land'. This hypothesis met strong opposition from at least one notable 
contemporary, Alfred R. Wallace (see text of letter to Hedley; Wallace 1974). 

In the most recent taxonomic review of the group (Haas 1935), species attributed to the 
genus Placostylus are found through some of the western Pacific Islands (New Zealand, 
New Caledonia, Solomon Islands, Fiji and Vanuatu). Haas (1935), who divided the genus 
into several subgenera, restricted the typical subgenus to New Caledonia (type species 
Limax fibratus Martyn, 1784; ICZN Opinion 1662 (ICZN 1992)). Several ‘subgenera’ are 
recognised that encompass the Pacific Island and New Zealand taxa, but the New Zealand- 
Lord Howe and New Caledonian (plus Loyalty Islands) taxa, with their large, solid shells, 
are more similar to one another than to the taxa further north, as noted by Hedley (1892). 
Because no other similar species are included in this grouping, it is reasonable to assume 
that the sister-taxon of the Lord Howe species is from either New Zealand, the Three Kings 
Islands or New Caledonia. 

The New Caledonian (including Loyalty Islands) Placostylus are still relatively poorly 
known, both taxonomically and biologically There are many names available in the 
literature, but no modern revisions have been published. Franc (1956) recognised 19 species 
but, using anatomical characteristics, Chérel-Mora (1982, 1983) reduced these to four, 
although most of her work remains unpublished. Dr E. Neubert, who is currently 
investigating the taxonomy and anatomy of the group in New Caledonia and the region, 
stated that, of approximately 140 available names, six valid species and approximately 20 
geographic subspecies can be recognised in New Caledonia (Neubert 2001). The shell 
morphology of some of the New Caledonian taxa is very similar to that of Lord Howe Island 
and New Zealand taxa. The taxon included in the present analysis is P. fibratus (Fig. 2E,F), 
the type species of Placostylus. Many of the New Caledonian taxa are threatened (Neubert 
2001) and some are still used for food. Salas et al. (1997) provide some data on the biology 
of P. fibratus. 

While the systematics of the Lord Howe species is poorly understood (see Appendix), a 
recent survey of extant populations has been performed (Ponder and Chapman 1999), as 
well as a preliminary study of the genetics (Colgan and Ponder 2001). 

Species similar to Lord Howe Island Placostylus occur in northern New Zealand (Powell 
1947, 19515, Choat and Schiel 1980; Triggs and Sherley 1993; Sherley 1996) and the Three 
Kings Islands (off northern-most New Zealand; Powell 19514, Brook and Laurenson 1992). 
Members of the subgenus Maoricolpus Haas, 1935 (type species Bulimus shongii 
(= hongii) Lesson, 1830) include the species found in northern New Zealand and Lord 
Howe Island. The Three Kings Islands species (P. bollonsi Suter, 1908) is placed in a 
monotypic subgenus, namely Basileostylus Haas, 1935. 

In New Zealand, there are three main taxa. Placostylus hongii (Fig. 24) has been 
recorded from sites between Whangaroa and Whangarei on the mainland Northland and on 
offshore islands between Whangaroa and Great Barrier Island (Powell 1979; Browne 1980; 
Brook and McArdle 1999). Placostylus ambagiosus Suter, 1906 (Fig. 2B,C), comprising 
ten named extant subspecies (Powell 1947, 19515), is located in the northern-most tip of 
the Northland Peninsula, where it is confined to tiny remnant populations. The third taxon, 
P. bollonsi (Fig. 2D), is confined to the Three Kings Islands that lie 60 km NW of Cape 
Reinga. This group is comprised of one large island and three small islands, all of which 
have colonies of this snail (Brook and Laurenson 1992). The biota of these islands also 


162 Molluscan Research W. Ponder et al. 


Fig. 2. Shells of Placostylus species (all material from the Australian Museum, Sydney, NSW; 
dimensions are maximum length). Images not to same scale. A, P. hongii (Lesson), Tauranga-Kawai Point, 
6 km N of Whanaki, Northland, New Zealand, C.114955, 77.6 mm. B, P. ambagiosus paraspiritus Powell, 
paratypes, headland 1 mile S of Cape Maria van Diemen, Northland, C.115002, 64.4 mm. C, 
P. ambagiosus whareana Powell, paratype, valley to north of Whareana Stream, between Waikuku Beach 
and Parengarenga, Northland, C.115001, 82 mm. D, P. bollonsi bollonsi (Suter), paratype, Big King 
Island, Three Kings Islands, C.29117, 91.5 mm. E,F, P. fibratus (Martyn), Isle of Pines, New Caledonia, 
C.409415, 84 mm (£) and Bourail, New Caledonia, C.409409, 84 mm (7). 


includes many other endemics. All the New Zealand species of Placostylus have received 
considerable taxonomic (Powell 1938, 1947, 1948, 1951a, 19515; Climo 1973; Sherley 
1996) and, more recently, conservation attention (Brook and Laurenson 1992; Triggs and 
Sherley 1993, Sherley 1996; Sherley et al. 1998), including the publication of a Recovery 
Plan (Parrish et al. 1995). 

Some of the New Zealand taxa are critically endangered: the Three Kings species, 
P. bollonsi, is confined to two small populations distinguished as separate subspecies 


Placostylus relationships based on mtDNA Molluscan Research 163 


(Powell 1948, 1951a; Climo 1973), one of approximately 130 individuals in a small area of 
only approximately 1.69 ha and the other of approximately 360 individuals in 
approximately 2.7 ha (Brook and Laurenson 1992). These populations are restricted by lack 
of suitable habitat and are not threatened significantly by predators. Placostylus 
ambagiosus, a species found only in far northern New Zealand, consists of a number of 
named subspecies (Powell 1938, 1947, 19515) confined to tiny remnant populations that 
show some degree of genetic structuring using isozyme electrophoresis (Triggs and Sherley 
1993). Some of these populations have fewer than 10 living individuals (Parrish ef al. 1995) 
and all are threatened by introduced predators (rats, birds and pigs). Placostylus hongii, 
which lives south of P. ambagiosus in Northland, is known from a few mainland locations 
and some offshore islands and is extant from five mainland populations and three offshore 
island groups (Brook and McArdle 1999). Translocation of the two mainland species has 
been undertaken for conservation purposes (Sherley 1994). 

The broader phylogenetic relationships of Placostylus within the Bulimulidae are still 
not fully understood. Solem (1959) argued for a relationship between Bothriembryon, the 
New Hebridean (i.e. Vanuatu) Diplomorpha and Placostylus based on anatomical data, 
with the latter genus having a northern origin. However, Breure (1979) placed Placostylus 
in a separate subfamily that he regarded as the sister-taxon to the rest of the family. He 
hypothesised that members of the Placostylinae reached New Zealand via east Antarctica 
and moved north. According to Breure (1979), placostylines show little relationship with 
the buliminids found in the western and southern parts of Australia (Bothrembrion; 
subfamily Orthalicinae). 

The present paper provides evidence from molecular data relating to the question of the 
origin of the Lord Howe Island Placostylus. Because the “subspecific” taxa (see Appendix) 
on Lord Howe Island are extinct, their status cannot be assessed using this methodology. 


Material and methods 


Material 


A list of the specimens sequenced is given in Table 1. The sequenced specimen of P. fibratus came from a 
population with intermediate characteristics between P. fibratus fibratus and P. fibratus souvillei Kobelt, 
1891 (Dr E. Neubert, personal communication). -- 


DNA extraction and sequencing 


Lord Howe Island and New Caledonia specimens were extracted and sequenced in Sydney, whereas New 
Zealand specimens were extracted and sequenced at Landcare Research, Auckland. 

For New Zealand specimens, foot muscle tissue was homogenised in 500 uL extraction buffer (0.01 M 
EDTA, 0.05 M NaCl, 0.5 M Tris-HCl, pH 8.0, 2% sodium dodecyl sulfate). A 10 uL aliquot of 10 mg mL `° 
! proteinase K (Boehringer, Mannheim, Germany) was added and the homogenate incubated at 65?C for 1 
h. Samples were extracted twice with phenol/chloroform/isoamyl alcohol (25:24: 1), followed by extraction 
with chloroform/isoamyl alcohol (24:1). The DNA was ethanol precipitated and the pellet rehydrated in 
50 uL buffer (10 mM Tris Cl (pH 7.4), 1 mM EDTA (pH 8.0)) following RNAse treatment. 

A 655 bp region of the cytochrome c oxidase 1 (COI) gene was amplified by polymerase chain reaction 
(PCR) using primers LCO-1490 (5”-GGTCAACA AATCATA AAGATATTGG-3 P) and HCO-2198 (5 - 
TA AACTTCAGGGTGACCAAAA A ATCA-3?) designed by Folmer et al. (1994). Amplifications were 
performed in a volume of 50 uL and consisted of 10 pmol of each primer, 10 mM Tris-Cl, pH 8.3, 1.5 mM 
MgCl, 50 mM KCl and 0.2 mM of each dNTP. The addition of 2 units Taq polymerase (Boehringer) 
followed an initial step of 2 min denaturation at 94°C. Cycling consisted of denaturation at 94°C for 1 min, 
annealing at 50°C (CO/) for 1 min and extension at 72°C for 1 min and 30 s, for 35 cycles. A final cycle | 
included a 5 min extension at 72?C. 

The PCR products were purified using the QIAquickTM PCR direct purification kit (Qiagen, Venlo, The 
Netherlands), according to the manufacturer’s instructions. Direct sequencing of purified products by the 


W. Ponder et al. 


Molluscan Research 


164 


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Placostylus relationships based on mtDNA Molluscan Research 165 


primers used for the original amplification was achieved using the Prism Ready Reaction Dye Deoxy 
Terminator Cycle Sequencing Kit (Applied Biosystems, Norwalk, CT, USA), following the manufacturer's 
instructions. Sequences were analysed on an automated DNA sequencer (model 377; Applied Biosystems). 
Two independent PCR products from each specimen were sequenced in both directions. 

For the Sydney experiments, molecular biological procedures generally followed the methods of Colgan 
et al. (2000a, 20005). The DNA extraction of foot muscle followed the hexadecyltrimethylammonium 
bromide procedure of Saghai-Maroof et al. (1984). 

The Sydney primer pair for CO/ is listed below with degeneracies written with standard TUB codes. 
Numbers in parentheses indicate the 5” end of the sequence in the complete mitochondrial genome of the 
earthworm Lumbricus terrestris (Boore and Brown 1995) COXIAFE (19) CWAATCAYA 
AAGATATTGGAAC; COX IAR, (726) AATATAWACTTCWGGGTGACC. Polymerase chain reaction 
cycling used the same basic protocols as Colgan et al. (2000a, 20002), with a general reaction mix of 1.0 
U Red Hot™ thermostable DNA polymerase, buffer IV (10x, 20 mM (NH4),SO,, 750 mM Tris-HCL, pH 
9.0, 0.196 (w/v) Tween; Advanced Biotechnologies, Leatherhead, Surrey, UK), 0.05 mM dNTPs, 2.5 mM 
MgCl,,12.5 pmol of each primer and 1 uL of a dilution (usually 1:10) DNA sample in a reaction volume 
of 50 uL overlaid with 30 uL oil. The usual cycling profile was denaturation at 95°C for 4 min, annealing 
at 45°C for 45 s and extension at 72°C for 30 s (one cycle), followed by 32 cycles of 95°C for 30 s, 45°C 
for 45 s and 72°C for 30 s and one cycle of 95°C for 30 s, 45°C for 45 s and 72°C for 5 min. The PCR 
products were purified using the QIAquickTM PCR Purification Kit and sequenced in both directions with 
an automated DNA sequencer (ABI? 310; Applied Biosystems) using the BigDye™ version 2.0 
sequencing kit. 


Alignment and sequence composition 


Sequences were edited using Sequence Navigator version 1.0.1 (Anonymous 1994). A compilation of all 
sequences was aligned using the default values for parameters in CLUSTAL W (Thompson ef al. 1994). It 
was not necessary to alter this alignment manually.-Data were compiled using MacClade (Maddison and 
Maddison 1992). 

Maximum parsimony was conducted in PAUP* 4.0b9 (Swofford 2000) with the default conditions for 
parsimony analyses with branch and bound searches guaranteed to find the shortest trees. All characteristics 
were unordered and unweighted. The steepest descent option was not enforced and accelerated 
transformation for character optimisation was assumed. Zero length branches were collapsed to give 
polytomies. Gaps were treated as unknown in all analyses. Bootstrap pseudo-sampling was conducted for 
100 replicates with branch and bound searching in each. Shortest trees satisfying alternative hypotheses of 
relationships were sought with the same strategy but using constraints in PAUP. 

For maximum likelihood analyses in PAUP*, a Hasegawa-Kishino-Yano (HKY) invariant model was 
used with the following settings. The number of substitution types was two, with the transition transversion 
ratio estimated by maximum likelihood. Empirical nucleotide frequencies were used. The proportion of 
invariable sites was estimated via likelihood and the distribution of rates at variable sites was assumed 
equal. The molecular clock model was not enforced. Other parameters took the default values in PAUP* 4. 
Comparison of trees using Kishino-Hasegawa likelihood tests assumed a normal approximation, with a 
two-tailed test. Shimodaira-Hasegawa tests used a one-tailed resampling of estimated log-likelihoods 
bootstrap with 1000 replicates. 

Outgroup sequences were obtained from the helicoidean pulmonates Aegista scepasma 
(Bradybaenidae) (Genbank Accession AB024900; Shimizu and Ueshima 2000), Cepaea nemoralis 
(Helicidae) (Genbank Accession U23045; Yamazaki ef al. 1997), Euhadra herklotsi (Bradybaenidae) 
(Genbank Accession Z71701; Yamazaki ef al. 1997) and Albinaria caerulea (Genbank Accession 
NC. 001761; Hatzoglou et al. 1995). 


Morphometrics 


Shell measurements were made using callipers. The discriminant analysis was undertaken using SYSTAT™ 
version 10 (SPSS, Chicago, IL, USA). 


Results 


The locations of samples are shown in Table 1. 
GenBank accession numbers for the CO/ sequences used are AY 165836—AY 165843, 
AY 165852 and AY290737—AY 290745. The alignment used in the analysis is available from 


166 Molluscan Research W. Ponder et al. 


the authors. As usual with mitochondrial sequences, there is a major AT bias in nucleotide 
composition. The mean overall percentages of nucleotides is as follows: A, 26.844; C, 
14.653; G, 17.174; T, 41.328 (overall AT average 68.17%). 

The probability that the nucleotide composition ofthe sequences is homogeneous is very 
close to 1 (x? = 37.600, d.f. = 63, P = 0.99), suggesting that variation in base composition 
between taxa has little effect on the analyses. 

The levels of pairwise difference of the HKY measure of genetic distance within the 
Placostylus samples vary between 0.0188 (within Lord Howe Island) and 0.6278 (between 
P. ambagiosus annectens and P bollonsi “north-east”) within New Zealand samples. In 
Table 2, the averages of the Kimura two-parameter distance are given for comparisons 
within and between various species. Within species, the averages are less than 0.1, but 
distances between species rise to 0.2014 for the comparison of Lord Howe Island snails 
with P. bollonsi. 

The results are illustrated in Figs 3, 4. The consensus of the maximum parsimony trees 
(Fig. 3) includes the following strongly supported monophyletic clades: Lord Howe Island 
specimens (with a decay index of 27), P. ambagiosus, P. ambagiosus + P. hongii and 
P bollonsi. Notably, the New Zealand samples are split into two distinct lineages. 
Constraining these to be monophyletic requires five more steps than the unconstrained, 
most parsimonious trees. The Lord Howe Island samples are sister to the P. ambagiosus + 
P. hongii clade. This group is, in turn, sister to the New Caledonian sample. Four extra steps 
are required to make the Lord Howe specimens sister to this to the exclusion of the New 
Zealand snails. A sister pairing of Lord Howe and New Caledonia specimens is seen in only 
6.6% of bootstrap trees compared with 38% of bootstrap replicates showing the pairing of 
Lord Howe Island with P. ambagiosus + P. hongii. The sister pairing of New Zealand 
P. ambagiosus + P. hongii with New Caledonia or all the New Zealand taxa with New 
Caledonia requires, respectively, two and five more steps than the maximum parsimony 
tree. Comparisons of the likelihoods of the trees satisfying various constraints are shown 
in Table 3 and the maximum likelihood tree is shown in Fig. 4. In particular, the Kishino— 
Hasegawa tests reject the possibility that there is a single New Zealand radiation that is 


Table 2. Average of pairwise genetic distances within and between 
Placostylus species using the Kimura two-parameter genetic distance 
= = omma c RR tame 

Comparisons Average 
—— — —— E  —  — `> 
Within species 


P. ambagiosus 0.0144 
P. bivaricosus 0.0630 
P. bollonsi 0.0028 
Between species 
P. ambagiosus/P. bivaricosus 0.1581 
P. ambagiosus/P. bollonsi 0.1755 
P. ambagiosus/P. fibratus 0.1345 
P. ambagiosus/P. hongii 0.0572 
P. bivaricosus/P. bollonsi 0.2014 
P. bivaricosus/P. fibratus 0.1717 
P. bivaricosus/P. hongii 0.1494 
P. bollonsi/P. fibratus 0.1849 
P. bollonsi/P. hongii 0.1732 
P. fibratus/P. hongii 0.1325 


————————————MÁ—ÓRÉÓÉÓÉÓÉÓR—€——— 


Placostylus relationships based on mtDNA 


100 
3 
100 
27 
3 
a 
100 | gg 
42 | 2 
R 96 
7 
70 
5 
53 
100 1 
14 


52 


Molluscan Research 


Aegista 
Cepaea 
Albinaria 
Euhaara 
LHI1 
LHI2 


LHI4 


Placostylus 
bivaricosus 
Lord Howe Island 


LHI5 


LHI6 


LHI7 


LHI8 


LHI9 


P. a. annectans 


Placostylus 
P. a. lesleyae ambagiosus 
North Cape area 
P.a. "irikavva" Northland 
P. a. watti 


Placostylus hongii Northland 
Placostylus fibratus New Caledonia 


P. b. arbutus 


Placostylus 
bollonsi 
Three Kings Islands 


P. b. bollonsi 
P. b. caperatus 


P. b. 'northeast' 


167 


Fig. 3. The strict consensus of 50 trees found in a branch and bound search of the part cytochrome c 
oxidase 1 gene. The trees were 556 steps long with a consistency index of 0.678. Bootstrap percentages 
above 50% are written above branches and Bremer decay indices below. The species names for Placostylus 


are indicated in bold. 


168 Molluscan Research W. Ponder et al. 


Table3. Comparisons of the likelihoods of trees constrained to show various relationships 


Tree -InL Diff —In L Kishino— Shimodaira— 
Hasegavva Hasegawa 

Unconstrained 3019.61928 

NC with NZ 3025.29525 5.67597 0.000* 0.182 

NC with LHI 3023.03754 3.41826 0.000* 0.346 

LHI with NC + part NZ 3023.02717 3.40789 0.000* 0.339 


l ii 0“ U e 
The first column shows the imposed constraint (if any), the second column shows the negative of the log 


likelihood of the maximum likelihood (ML) trees, the third column shows the difference between tree 
likelihoods and the last two columns give the probabilities that the compared trees have the same 
likelihood under the Kishino-Hasegawa (two-tailed) test and the Shimodaira-Hasegawa (one-tailed) 
test. The constraints are that: (/) New Caledonia (NC) and all New Zealand (NZ) specimens are a sister 
pair; (2) New Caledonia and all Lord Howe Island (LHI) snails form a sister pair; and (3) the Lord Howe 
Island snails are sister to a group comprising the New Caledonian sample and New Zealand P. hongii 
and P. ambagiosus. 


sister to the New Caledonian sample (constraint 1) or that this latter specimen and the Lord 
Howe Island snails are sister taxa (constraint 2). 


Discussion 


New Caledonia and New Zealand were separated by the end of the Cretaceous (Hall 2002), 
but it is unclear as to how much of the Norfolk Ridge remained emergent through the early 
Tertiary. The oldest basalts on Lord Howe Island were formed only 6.4 million years ago. 
Thus, the geological evidence indicates that dispersal, rather than vicariance, must account 
for the presence of Placostylus on Lord Howe Island. While we can only speculate about 
possible dispersal mechanisms, an analysis such as this can provide evidence for the likely 
origin of the source for the dispersal event. This does not eliminate the probability that past 
intermediate populations, for example, on islands that are now submerged seamounts, 
ridges or plateaus to the north of Lord Howe Island (or the Three Kings Islands), may have 
facilitated dispersal as “stepping stones”. 

The Three Kings Islands are comprised of indurated, deformed marine sedimentary and 
volcanic rocks of probable early Cretaceous age (Haywood and Moore 1987; Brook and 
Laurenson 1992). Brook and Laurenson (1992) state that “... it is possible that marine 
conditions persisted in the vicinity of present day Three Kings Islands for much or all of 
that time” (Cretaceous to early Caenozoic). The present configuration of northern New 
Zealand (including the Three Kings) was probably developed during the mid-Caenozoic, 
with a subaerial peninsula developed from about Kaitaia/Doubtless Bay to the Three Kings 
area in the early Miocene (15-20 million years ago), this degenerating to a few islands by 
the mid-late Miocene (Brook and Thrasher 1991). The northern-most end of Northland 
(*North Cape") was, at that time, a separate island and was connected to the rest of 
Northland subsequent to the early Pliocene (5-3 million years ago; Pillans et al. 1992). 

Whereas dispersal must be invoked to account for the presence of Placostylus on Lord 
Howe Island, the Three Kings Islands could, seemingly, have been connected or nearly 
connected to Northland in the mid-Tertiary and closely adjacent (approximately 10 km) 
during interglacial periods in the Pleistocene (Brook and Laurenson 1992). Given these 
data, postulating an origin of P. bollonsi from Northland seems logical and has been argued 
for by Brook and Laurenson (1992). However, there is little support for this hypothesis from 
our data, which suggest that the Three Kings taxon and the mainland taxa represent two 


Placostylus relationships based on mtDNA Molluscan Research 169 


Aegista 
Cepaea 
Euhadra 
Albinaria 
LHI1 
LHI2 
LHI4 
LHI5 
LHI8 
LHI9 
LHI6 
LHI7 
P. a. annectans 
P. a. watti 
P. a. lesleyae 
P.a. 'tirikawa' 
P. hongii 
P. fibratus 
P. b. arbutus 


P. b. bollonsi 


P. b. caperatus 


P. b. 'northeast' 
—— 10 changes 


Fig.4. Phylogram for the cytochrome c oxidase 1 data of the maximum likelihood tree resulting from 
an heuristic search with 10 replicates of random sequence additions. See Fig. 3 for details of taxa. 


separate clades, as reflected in the allocation of different subgeneric names. It appears that 
the Three Kings taxon is the result of either a long overseas dispersal event or (more 
probably, in our opinion) it represents a surviving relict of a taxon distributed on now 
submerged islands along the Norfolk Ridge. 

Our results clearly suggest a sister relationship between the northland P. ambagiosus and 
P. hongii, separated by the late Pliocene isthmus between what was once a northern island. 
Whereas there is strong support for the monophyly of each of the main radiations 
(P. ambagiosus, P. hongii; P. bollonsi and P. bivaricosus), regrettably, the analysis IS 
weakened by there being only a single sample from New Caledonia. 

Brook and McArdle (1999) have advocated pre-European Maori transport of 
Placostylus hongii (which was used as a food item; Haywood and Brook 1981) to and from 
offshore islands to account for their present distribution. There is no evidence of pre- 
European contact with Lord Howe Island, so some other form of overseas dispersal must 
have occurred. This most likely occurred as a result of an individual being carried in a 


170 Molluscan Research W. Ponder et al. 


severe storm to the island. Whereas this may have come from a nearby population on an 
island (that has since disappeared) to the north, given that there is a chain of seamounts 
northwards, the results of our analysis suggest that there was a common origin with the 
mainland New Zealand taxa. Self-fertilisation is common in pulmonate snails, so even a 
single juvenile could establish a population. Placostylus hatchlings are known to be often 
arboreal, this habit facilitating such a mode of dispersal of this otherwise ground-dwelling 
species. Extremely rare aerial dispersal of hatchlings and juveniles, less than 1 cm in length, 
during major storms seems, to us, to be a more feasible explanation of dispersal than other 
hypotheses invoking rafting or transport by birds. 

The results show low levels of genetic divergence between individual Lord Howe Island 
sequences (Fig. 3) but, on the available small amount of material, no clear pattern emerges 
(16S rDNA sequences are also available for the Lord Howe Island samples and show 
substructure between the available populations (Colgan and Ponder 2001)) There is 
considerable variation in shell morphology within the area containing the material used in 
the analysis (Ponder and Chapman 1999) but, again, the material sampled is not adequate 
to address questions relating to whether there is any underlying genetic basis for this 
variation. The range of the available samples from Lord Howe Island does not cover all the 
island, with none coming from the hills and mountains in the southern part where 
Placostylus appears to be extinct. While it remains possible that more southerly 
populations, from the remote areas around Mounts Lidgbird and Gower, may still be extant, 
it remains untested whether they are genetically distinct, as suggested by their shell 
morphology, or whether there is a significant geographic barrier to gene flow in this region. 
Triggs and Sherley (1993) showed that there was a considerable level of genetic variation 
between populations of P. ambagiosus. In these cases, small but overlapping differences in 
shell morphology led to prior creation of several subspecies (Powell 1938, 1947, 1948, 
19515; Sherley 1996). 

The genetic status of New Zealand Placostylus species has been assessed by allozyme 
electrophoresis (Triggs and Sherley 1993). Evidence of distinct lineages (possibly at the 
species level) was found in one mainland morphospecies, P. ambagiosus, over a geographic 
scale of tens of kilometres, with differentiation apparently fostered by Pleistocene and late 
Pliocene sea level changes. These authors found no significant variation in allozymes in 
P. bollonsi from the Three Kings Islands, but the results presented herein suggest that at 
least the north-east population may be distinct. 

The results clearly discount the Lord Howe Island taxon being a recent introduction from 
New Zealand but, because we have only one specimen from New Caledonia, where there 
are probably six Recent species present, the possibility of a relatively recent introduction 
from that source cannot be discounted. 


Conservation status of Lord Howe Island Placostylus 


Etheridge (1889) noted that P/acostylus on Lord Howe Island was *... found everywhere 
under cover, and in immense numbers’ and that it “... appears to avoid open spaces as a rule, 
and prefers shady damp situations and the scrubby hill sides where composed of the Coral- 
sand rock. It is sparingly represented even at the higher altitudes, being reported as seen 
under the ‘wall’ of Mount Ledgbird.’ Hedley (1891) noted that it was °... all over the island 
in sheltered places under stones; abundant’. 

However, this previously abundant animal has been the subject of conservation concern 
for some time. Iredale (1944) noted that there was a “... new difficulty of recent years, the 
destruction effected by the rat plague, written up by Hindwood'. More recently, Smithers 


Placostylus relationships based on mtDNA Molluscan Research 171 


et al. (1977) noted that Placostylus is apparently “... existing in only four small colonies, 
whereas from early reports it was dispersed widely from sea-level up to the mountain tops”. 
Ponder and Chapman (1999) reported that all the living specimens found in their 1999 
survey were located on low, rather flat ground with ample litter. In most cases, the samples 
were taken on calcarenite soils or sandy soils derived, in large part, from calcarenite. Living 
specimens were found under well-developed litter. Placostylus seem to prefer the vicinity 
of the large native fig trees, but not exclusively so. 

Placostylus bivaricosus is listed as endangered in NSW under the Threatened Species 
Conservation Act and a Recovery Plan (NSW National Parks and Wildlife Service 2000) 
has been prepared. 


Acknowledgments 


The collection of the New Caledonian P/acostylus was arranged by Dr P. Bouchet, 
facilitated by Bertrand Richer De Forges and collected by Alain Lapetite. Pam Da Costa 
sequenced this sample. Michael Murphy, Dean Hiscox and Lisa Menke assisted with the 
collection of material on Lord Howe Island and the Lord Howe Island Board gave 
permission to undertake the work. DMG and GHS thank Richard Parrish from the 
Department of Conservation (New Zealand) for field assistance. Joshua Studdert assisted 
with preparing the map and the plates and with photographing specimens. Holly Barlow 
and Joshua Studdert took the measurements used in the shell comparisons. 


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Placostylus relationships based on mtDNA Molluscan Research 175 


Appendix. The Lord Howe Island Placostylus taxa 


Placostylus bivaricosus (Gaskoin, 1855) is the only currently recognised species found on 
Lord Howe Island (Smith 1992). The last review of this group was by Iredale (1944), who 
noted that the: *variation in this group is worthy of study ... At the present time the various 
forms may be less visible in the field than in Roy Bell's time, when he indicated no less than 
six different colonies, separable in the field’. 

No critical examination of the taxonomy of the Lord Howe Island Placostylus has ever 
been undertaken, although Smith (1992) catalogued the taxa and details of original names 
and references can be obtained from that source. Material in the Australian Museum 
collections appears to support the recognition of at least three, possibly four (see below), 
Recent and one fossil taxon on the basis of shell morphology (Ponder and Chapman 1999). 
These can tentatively be treated as “subspecies” as follows. 


Placostylus bivaricosus bivaricosus (Gaskoin, 1855) 


Bulimus bivaricosus Gaskoin, 1855. Lord Howe Island (location of type material not known; fide 
Smith 1992: 108). 

Placostylus bivaricosus belli Iredale, 1944. Lord Howe Island (holotype Australian Museum 
C.38973; Fig. 5C). 


Remarks 


The typical subspecies (Fig. 54,3) is found on the northern end of the island and in the 
settlement area as far south as Intermediate Hill. Placostylus bivaricosus belli (Fig. 5C) is 
based on a small, narrow (shell length 54.6 mm, width 22.2 mm, aperture length 24.3 mm) 
specimen that can be matched with other occasional small specimens found within the 
settlement area. 


Placostylus bivaricosus etheridgei (Hedley, 1891) 
Bulimus bivaricosus etheridgei Hedley, 1891. Under the wall of Mt Lidgbird (Hedley, 1891). Iredale 
(1944) states that this species is “apparently from the saddle of Mt Lidgbird' (three syntypes, 


Australian Museum C.33308; Fig. 5H).- : 
Placostylus etheridgei ‘Brazier’, 1889. Nomen nudum (introduced as a name on the caption to a figure 


in Etheridge, 1889). 
Placostylus bivaricosus royi Iredale, 1944. Little Slope, base of eastern side of Mount Gower 


(holotype, Australian Museum C.38974; Fig. 51). 


Remarks 


This taxon is found in the southern mountains. It is variable in size, although often large, 
and has a thin shell with a weakly developed apertural varix and the surface of the shell is 
sculptured with oblique rugae. It is probably extinct, no living specimens having been 
collected for several decades. There is some suggestion from museum material of overlap, 
or perhaps merging, of typical bivaricosus and etheridgei in the area around Intermediate 
Hill. Unfortunately, the populations in this area are now either extremely reduced or, more 
likely, extinct. Both the available names for this taxon are from populations around the base 
of Mount Lidgbird and Mount Gower, or in the Erskine Valley, which lies between the two 
mountains. These have a significantly larger shell than samples from the top of Mount 
Gower (Fig. 5D,F; see below). 


176 Molluscan Research W. Ponder et al. 


Fig.5. Shells of Placostylus from Lord Howe Island (all material from the Australian Museum, Sydney, 
NSW; dimensions are maximum length). Images not to same scale. 4,5, P. bivaricosus bivaricosus 
(Gaskoin), Lord Howe Island, C.114669, 56.6 mm (A) and C.114666, 60.7 mm (B). C, Holotype of P. 
bivaricosus belli Iredale, Lord Howe Island, C.38973, 54.6 mm. D,F, P. aff. bivaricosus etheridgei 
(Hedley), Mount Gower (C.114677), 54.0 mm (D) and 62.2 mm (F). E, P. bivaricosus cuniculinsulae 
(Cox), Rabbit (= Blackburn) Island, Lord Howe Island, C.118151, 45.6 mm. G, P. bivaricosus solidus 
(Etheridge), syntype, Lord Howe Island, C.171111, 74.8 mm. HI, P. bivaricosus etheridgei (Hedley), 
syntype of Bulinus etheridgei Hedley, Mt Lidgbird, Lord Howe Island, C.33308, 67.2 mm (H) and 
holotype of P. bivaricosus royi Iredale, Little Slope, Lord Howe Island, C.38974, 77.2 mm (1). 


Placostylus relationships based on mtDNA Molluscan Research 177 


Placostylus bivaricosus cuniculinsulae (Cox, 1872) 


Bulimus (Placostylus) cuniculinsulae Cox, 1872. Rabbit Island (= Blackburn Island), Lord Howe 
Island (holotype, Natural History Museum, London, 1888.12.1 1.47-8). 


Remarks 


This small form was found only on Blackburn Island and is now extinct (Iredale 1944). 
Etheridge (1889) noted that there was a great deal of variation in shell morphology and that 
it seemed “... more in keeping with the facts to regard the Rabbit Island shell simply as a 
variety'. However, the material in the Australian Museum from Blackburn Island all 
possesses a distinctive, thin shell and is smaller than any adults seen from the main island 
(Figs 5E, 6; Table 4). 


Placostylus bivaricosus solidus (Brazier in Etheridge, 1889) 


Bulimus bivaricosus solida Brazier in Etheridge, 1889. (Seven syntypes, Australian Museum 
C.171111 (Fig. 5G), C.171110). 


Remarks 


This large, heavy form is found in calcarenite along the eastern side of the settlement area 
of the island. Smith (1992) indicates that the original introduction of the name is a nomen 
nudum. However, there is a statement differentiating the taxon in the original introduction 
of the name (p. 27), so it is valid. In addition, Etheridge (1891) described this taxon in 
greater detail and provided figures. There are also fossil specimens that appear to be 
indistinguishable from the Recent material (including one of the syntypes in C.171111), 
although specimens can usually be readily separated into the solida morph or the Recent 
morph. Unfortunately, there has not yet been any attempt to systematically collect fossil 
Placostylus and relate shell morphology to stratigraphy. 

A comparison of three basic shell measurements (Table 4) indicates that the Recent taxa 
discriminate well (Fig. 6), but mainly on size rather than shape differences. There also 
appears to be justification for the recognition of an additional unnamed and recently extinct 
taxon that lived on the top of Mount Gower (Australian Museum C.I 14677; Fig. 5D,F) and 
on a razor-back spur to the south of the summit (Australian Museum C.114679). These two 
lots have similar shell dimensions to typical P. bivaricosus (Fig. 6; Table 4), but have the 


Fig.6. Plotofthe first and second discriminant 
scores from the measurement data summarised in 
Table 4. The ellipses have a confidence level of 
P = 0.683. (V), Placostylus bivaricosus 
cuniculinsulae; (+), P. bivaricosus bivaricosus; 
(©), P. bivaricosus etheridgei; (O), P. bivaricosus 
aff. etheridgei (top of Mt Gower); (x), 

P. bivaricosus solidus. 


Score 2 


178 Molluscan Research 


W. Ponder et al. 


Table 4. Shell measurements of Placostylus from Lord Howe Island 


Data show the range and mean + s.d. 


e—a 


Shell length (mm) 


P. bivaricosus bivaricosus (n = 40) 
P. bivaricosus etheridgei (n = 19) 

P. bivaricosus cuniculinsulae (n ^ 9) 
Summit of Mount Gower (n = 11) 

P. bivaricosus solidus (n = 15) 


46.0-61.9, 53.4 + 3.5 
65.5-77.1, 71.5 x 4.1 
40.0-45.7, 43.3 + 1.8 
51.9-65.1, 57.6 + 4.2 
61.1—79.0, 71.4 + 6.2 


Shell width (mm) 


21.3-27.5, 23.8 + 1.5 
28.0-35.3, 31.9 + 2.3 
19.4-21.9, 20.3 + 0.8 
23.7-27.8,25.4 + 1.2 
26.8-36.6, 32.6 + 3.1 


Aperture length (mm) 


23.9-32.4, 26.9 € 2.1 
31.8-41.1,35.9 + 2.9 
20.3-22.9, 21.4 + 0.9 
26.5-31.9,28.3 + 1.8 
31.5-41.0, 37.2 € 2.7 


Specimens measured: P 5. bivaricosus (C118724; C114653; C.78759; C.114656); P. b. solidus (C.171111; C.171110; C.114648; 
C.47513); P. b. etheridgei (C.33308; C.38973; C.114672; C.114675); P. b. cuniculinsulae (C.118151; C.114646; C.114647); 


Mount Gower (C.114679; C.114677). 


Aperture length is the external, not internal, length. 


colour and surface sculpture of P. bivaricosus etheridgei. Given their isolation on the 
summit, they were probably genetically distinct from the populations around the base of the 


southern mountains and the typical lowland form. 


http://www.publish.csiro.au/journals/mr 


CSIRO PUBLISHING 
ÇO T ROLE edu 7 — — a 
www.publish.csiro.au/journals/mr Molluscan Research, 2003, 23, 179-183 


Short contribution 


Changes in tissue composition during larval development of the 
blacklip pearl oyster, Pinctada margaritifera (L.) 


Jan M. Strugnell^P€ and Paul C. Southgate^ 


Agchool of Marine Biology and Aquaculture, James Cook University, Townsville, Qld 4811, Australia. 
BMolecular Evolution, Department of Zoology, Oxford University, South Parks Rd, Oxford OX1 3PS, UK. 
CTo whom correspondence should be addressed. Email: jan.strugnell@merton.oxford.ac.uk 


Abstract 


This paper reports on the changes in proximate composition (i.e. protein, lipid and carbohydrate) of tropical 
blacklip pearl oyster Pinctada margaritifera (L., 1758), larvae throughout development. Protein was the 
largest component of dried larval tissues. Mean protein, lipid and carbohydrate contents all decreased from 
Day 1 to Day 4. Lipid loss between Day 1 and Day 4 contributed 56% of the total energy utilised during 
this period, whereas protein contributed almost 40%. Between Day 18 and Day 21, the accumulation of 
lipid contributed almost 7096 of the total energy gain per larva during this period, suggesting that lipid may 
be the primary energy reserve utilised during metamorphosis. Patterns of energy reserve composition, 
utilisation and accumulation within P. margaritifera larvae were comparable to those reported for temperate 


species. 


Introduction 

Fundamental differences exist between tropical and temperate marine environments that 
directly influence the organisms living within them. Tropical marine environments are 
characterised by surface-water temperatures of 20—30*C, relatively low nutrient loads and 
correspondingly low phytoplankton levels (Nybakken 1982). In contrast, temperate marine 
environments are characterised by seasonal, but relatively high, nutrient loads, high 
phytoplankton levels and water temperatures fluctuating between 10 and 20°C in a seasonal 
fashion (Nybakken 1982). On this basis, the rates of energy-metabolism in temperate 
bivalve molluscs cannot be assumed to pertain to tropical species. However, studies 
reporting on changes in proximate composition during larval development of bivalves have 
focused on temperate species (Holland and Spencer 1973; Bayne ef al. 1975; Gallager and 
Mann 1986; Gallager et al. 1986; Whyte et al. 1987, 1990, 1991). The hypothesis tested in 
this study was that patterns of energy-reserve utilisation and accumulation in larvae of the 
tropical blacklip pearl oyster, Pinctada margaritifera (L., 1758), differ from those reported 
for temperate species. This was examined by determining changes in proximate 
composition (i.e. protein, lipid and carbohydrate) during larval development. 


Materials and methods 


Spawning induction and larval rearing of P. margaritifera followed standard procedures (Southgate and 
Beer 1997). Ten hatchery-conditioned P. margaritifera broodstock were induced to spawn using thermal 
shock. Progeny from each of the females were randomly distributed across larval batches. Larvae were 
reared in six 500-L fibreglass tanks and water was changed every 48 h. Larvae were fed a diet consisting 
of the two prymnesiophytes, Pavlova salina and Isochrysis aff. galbana clone T-ISO, and the diatom 
Chaetoceros muelleri (Southgate and Beer 1997). A sample of approximately 15000 larvae was taken for 


© Malacological Society of Australasia 2003 10.1071/MR02018 1323-5818/03/020179 


180 Molluscan Research J. M. Strugnell and P. C. Southgate 


proximate analysis at each water change from each tank and stored in liquid nitrogen to await analysis of 
protein, lipid and carbohydrate content (Mann and Gallager 1985; Baethgen and Alley 1989). Prior to 
analysis, larval samples were freeze-dried. The experiment ended after 21 days, when 5096 of larvae were 
“eyed”. Water temperature was measured at 0800 hours and 1800 hours each day and ranged from 25?C to 
29?C with a mean of 27.2?C. 

Proximate compositional data were used to calculate total energy values using caloric equivalents of 
36.42, 23.86 and 17.16 kJ g ! for lipid, protein and carbohydrate respectively (Brett and Groves 1979). 


Results and discussion 


Pinctada margaritifera larvae increased in mean (+ SEM, n = 30) shell length from 76 + 
l um on Day 1 (24 h after fertilisation) to 213 + 5 um on Day 21. Mean (+ SEM, n = 30) 
larval dry weight increased from 556.6 + 1.1 ng larva! on Day 1 to 2793.4 + 40.0 ng larva! 
on Day 21. Mean survival of larvae to Day 21 was 13.2 + 5.096. 

Changes in mean protein, lipid and carbohydrate content of P. margaritifera larvae are 
shown in Fig. 1. Protein was the largest component of dried larval tissues. Mean protein 
content almost halved from 133.7 + 5.1 ng larva! on Day 1 to 67.3 + 3.1 ng larva”! on Day 
4 (+ SEM, n = 6). The most notable increase in mean protein content of larval tissues 
occurred between Day 12 (133.5 + 17.7 ng larva !) and Day 18 (395.2 + 12.9 ng larva !). 
Subsequently, the increase in protein content slowed considerably; the tissues of 21-day-old 
larvae possessed 417.7 + 16.78 ng larva ! of protein. 

Lipid was the second largest component of dried larval tissue during development. Mean 
lipid content decreased by greater than 75% from 73.3 + 9.1 ng larva! to 11.7 + 1.1 ng 
larva! between Day 1 and Day 4. Subsequently, larval lipid content increased to 96.8 + 
11.6 ng larva! on Day 15, declined to 76.9 + 5.2 ng larva! on Day 18, then increased 
rapidly to 140.6 + 2.2 ng larva! on Day 21. 

Carbohydrate was the smallest component of dried larval tissues. Mean carbohydrate 
content decreased by about 80% between Day 1 (13.5 + 4.9 ng larva ') and Day 4 (2.7 + 
0.8 ng larva !). Subsequently, carbohydrate content increased to 63.1 + 31.1 ng larva! on 
Day 21. 

Lipid loss between Day 1 and Day 4 contributed 56% of the total energy utilised during 
this period, whereas protein contributed almost 40%. The energy contributed by 
carbohydrate was very small, less than 10% of the amount contributed by lipid. Between 
Day 18 and Day 21, the accumulation of lipid contributed almost 7096 of the total energy 
gain per larva during this period. The energy available from lipid was more than four times 
that available from protein and carbohydrate, each of which contributed about 1596 of the 
total energy value at this time. 

Patterns of energy reserve composition, utilisation and accumulation within 
P. margaritifera larvae were found to be comparable to those reported for temperate species 
(Holland and Spencer 1973; Gallager et al. 1986; Whyte et al. 1987). As reported in all 
previous studies with bivalves (Holland 1978; Whyte et al. 1989), protein was the largest 
organic component of P. margaritifera larvae and showed the largest increase during larval 
development. Protein forms the bulk of the structural organic components of larval tissues 
(Holland 1978; Whyte et al. 1989). Also in accordance with previous studies, lipid was the 
second largest organic component of P. margaritifera larvae and carbohydrate content was 
relatively small and changed little during larval development. 

The marked decline in protein, lipid and carbohydrate content of P. margaritifera larvae 
between Days 1 and 4 is similar to that reported for the larvae of temperate scallops 
(Patinopecten yessoensis and Crassadoma gigantea), where protein, lipid and carbohydrate 
were reported to be catabolised simultaneously and linearly with time during embryonic 


Tissue composition of pearl oyster larvae Molluscan Research 181 


500 


---- Carbohydrate 

— Lipid prT 
= /F q 

— - - Protein 


400 


Tissue composition (ng larva”1) 


Age (days from fertilisation) 
Fig. 1. Changes in mean (+ SEM) protein, lipid and carbohydrate content of 
P. margaritifera larvae (ng larva!) during development (n = 6, Days 1 to 9; n = 3, Days 
12 to 21). 


development (Whyte ef al. 1990, 1991). This decline is thought to result from utilisation of 
endogenous reserves, provided to the egg by the female parent, to fuel the transition to an 
exogenous mode of feeding (Bayne et al. 1975; Mann and Gallager 1985). Utilisation of 
protein, lipid and carbohydrate by P. margaritifera between Days 1 and 4 shows that larval 
development during this period is an energetically expensive process; approximately 66% 
of the total energy content of 1-day-old larvae was utilised by Day 4. Similarly, 56.996 of 
the energy content of fertilised eggs was reported to be utilised during the first 72 h after 
fertilisation in scallop (Crassodoma gigantea) larvae (Whyte et al. 1990). 

Over half the energy utilised by P. margaritifera larvae between Days 1 and 4 was 
contributed by lipid, suggesting that lipid is the primary energy reserve during this period. 
Similarly, embryogenesis of the oyster (Crassostrea virginica) and clam (Mercenaria 
mercenaria) has been reported to be fuelled primarily (55-96% and 50-6576 respectively) 
by parentally derived lipid (Gallager and Mann 1986). 

A number of studies with temperate bivalves only investigated changes in the lipid 
content of tissues during larval development and disregarded changes in protein or 
carbohydrate contents (Gallager and Mann 1986; Gallager ef al. 1986; Napolitano et al. 
1988; Delaunay et al. 1992, 1993). However, in P. margaritifera larvae, protein was found 
to contribute 40% of the total energy utilised between Days 1 and 4, indicating it to be an 
important secondary energy source during this period. Similarly, Whyte et al. (1990, 1991) 
reported protein to contribute a significant portion of the total energy expended during the 
embryogenesis of the scallops, Patinopecten yessoensis and Crassodoma gigantea (44.9% 
and 43.5% respectively). Such values were only slightly lower than the energy contribution 
reported for lipid of 47.6% and 4% respectively (Whyte et al. 1990, 1991). 

Although the overall energy contribution from carbohydrate in P. margaritifera tissues 
was small between Days 1 and 4, it accounted for an 80% depletion of its initial energy 
content, which is comparable to the decline in lipid (8670). Similar depletion of 
carbohydrate has been reported during embryogenesis of scallops (Whyte et al. 1990, 
1991). In comparison, the decline in protein content of P. margaritifera larvae between 
Days 1 and 4 accounted for only 50% of the initial energy content. This reflects the 
important structural role of proteins in keeping the larval body intact (Holland 1978). 


182 Molluscan Research J. M. Strugnell and P. C. Southgate 


Larval lipid and carbohydrate contents increased markedly between Days 18 and 21. 
During the same period, the rate of protein accumulation declined considerably. 
Although both lipid and carbohydrate contents increased by almost 50% between Days 
18 and 21, accumulation of lipid contributed more than four times the total energy gain 
(per larva) than either protein or carbohydrate. This strongly suggests that lipid may be 
the primary energy reserve utilised during metamorphosis. Although this assumption is 
supported by the results of similar studies with temperate species (Holland and Spencer 
1973; Whyte et al. 1987), further study is required to confirm this role for lipid in P. 
margaritifera. 

These results provide a strong basis for future study by clearly indicating changes in 
proximate composition and energy-reserve utilisation and accumulation during larval 
development of P. margaritifera larvae. This research has increased our understanding of 
the energetics of bivalve larvae, being the first study of its kind to investigate a tropical 
species. These findings have significant practical application and will be useful in the 
development of more efficient hatchery techniques for pearl oysters; for example, in 
formulating diets that best provide the biochemical requirements of larvae to maximise 
growth and survival and to minimise the time to metamorphosis. 


References 


Baethgen, W. E., and Alley, M. N. (1989). A manual colorimetric procedure for measuring ammonium 
nitrogen in soil and plant Kjeldahl digests. Communications in Soil Science and Plant Analysis 20, 
961—969. 

Bayne, B. L., Gabbott, P. A., and Widdows, J. (1975). Some effects of stress in the adult on the eggs and 
larvae of Mytilus edulis L. Journal of the Marine Biological Association of the United Kingdom 55, 
675—689. 

Brett, J. R., and Groves, T. D. (1979). Physiological energetics. In “Fish Physiology, Vol. III’. (Eds W. S. 
Hoar and D. J. Randall.) pp. 279—351. (Academic Press: New York, USA.) 

Delaunay, F., Marty, Y., Moal, J., and Samain, J. F. (1992). Growth and lipid class composition of Pecten 
maximus (L.) larvae grown under hatchery conditions. Journal of Experimental Marine Biology and 
Ecology 163, 209-219. 

Delaunay, F., Marty, Y., Moal, J., and Samain, J. F. (1993). The effects of monospecific algal diets on growth 
and fatty acid composition of Pecten maximus (L.) larvae. Journal of Experimental Marine Biology and 
Ecology 173, 163-179. ; 

Gallager, S. M., and Mann, R. (1986). Growth and survival of larvae of Mercenaria mercenaria (L.) and 
Crassostrea virginica (Gmelin) relative to broodstock conditioning and lipid content of eggs. 
Aquaculture 56, 105—121. 

Gallager, S. M., Mann R., and Sasaki, G. C. (1986). Lipid as an index of growth and viability in three 
species of bivalve larvae. Aquaculture 56, 81-103. 

Holland, D. L. (1978). Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. 
In “Biochemical and Biophysical Perspectives in Marine Biology”. (Eds P. C. Malins and J. R. Sargent.) 
pp. 85-123. (Academic Press: London, UK.) 

Holland, D. L., and Spencer, B. E. (1973). Biochemical changes in fed and starved oysters, Ostrea edulis L. 
during larval development, metamorphosis and early spat growth. Journal of the Marine Biological 
Association of the United Kingdom 53, 287—298. 

Mann, R., and Gallager, S. M. (1985). Physiological and biochemical energetics of larvae of Teredo navalis 
L., and Bankia gouldi (Bartsch) (Bivalvia: Teredinidae). Journal of Experimental Marine Biology and 
Ecology 85, 211-228. 

Napolitano, G. E., Ratnayake, W. M. N., and Ackman, R. G. (1988). Fatty acid components of larval Ostrea 
edulis (L.); importance of triacylglycerols as a fatty acid reserve. Comparative Biochemistry and 
Physiology 90B(4), 8975—883. 

Nybakken, J. W. (1982). “Marine Biology: An Ecological Approach.’ (Harper and Row: New York, USA.) 

Southgate, P. C., and Beer, A. C. (1997). Hatchery and early nursery culture of the blacklip pearl oyster 
(Pinctada margaritifera L.). Journal of Shellfish Research 16(2), 561—567. 


Tissue composition of pearl oyster larvae Molluscan Research 183 


Whyte, J. N. C., Bourne, N., and Hodgson, C. A. (1987). Assessment of biochemical composition and 
energy reserves in larvae of the scallop Patinopecten yessoensis. Journal of Experimental Marine 
Biology and Ecology 113, 113—124. 

Whyte, J. N. C., Bourne, N., and Hodgson, C. A. (1989). Influence of algal diets on biochemical 
composition and energy reserves in Patinopecten yessoensis (Jay) larvae. Aquaculture 78, 333-347. 
Whyte, J. N. C., Bourne, N., and Ginther, N. G. (1990). Biochemical and energy changes during 

embryogenesis in the rock scallop Crassodoma gigantea. Marine Biology 106, 239—244. 

VVhyte, J. N. C., Bourne, N., and Ginther, N. G. (1991). Depletion of nutrient reserves during embryogenesis 
in the scallop Patinopecten yessoensis (Jay). Journal of Experimental Marine Biology and Ecology 149, 
67-79. 


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Molluscan 
Research 


Contents 
Volume 23 Number 2 2003 


Embryonic and larval development of Pinctada margaritifera (Linnaeus, 1758) 
M. S. Doroudi and P. C. Southgate 101 


Ultrastructure of male germ cells in the testes of abalone, Haliotis ovina Gmelin 
S. Singhakaew, V. Seehabutr, M. Kruatrachue, P. Sretarugsa and S. Romratanapun 109 


Gastropod phylogeny based on six segments from four genes representing coding 
or non-coding and mitochondrial or nuclear DNA 
D. J. Colgan, W. F. Ponder, E. Beacham and J. M. Macaranas 123 


Reassessment of Australia's oldest freshwater snail, Viviparus (?) albascopularis 
Etheridge, 1902 (Mollusca : Gastropoda: Viviparidae), from the Lower 
Cretaceous (Aptian, Wallumbilla Formation) of White Cliffs, New South Wales 
B. P. Kear, R. J. Hamilton-Bruce, B. J. Smith and K. L. Gowlett-Holmes 149 


Relationships of Placostylus from Lord Howe Island: an investigation using the 
mitochondrial cytochrome c oxidase 1 gene 
W. F. Ponder, D. J. Colgan, D. M. Gleeson and G. H. Sherley 159 


Short contribution 


Changes in tissue composition during larval development of the blacklip 
pearl oyster, Pinctada margaritifera (L.) 
J. M. Strugnell and P. C. Southgate 179 


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